Improved bacterial endotoxin test for the determination of endotoxins

ABSTRACT

Herein is reported a method for determining bacterial endotoxin at low concentrations in a sample of an antibody (that has been produced using bacterial cells) comprising the following steps in the following order: i) adding magnesium ions to the sample, ii) diluting the sample, iii) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution, and iv) determining bacterial endotoxin in the sample using a bacterial endotoxin test, particularly the  limulus  amoebocyte lysate as say.

Herein reported is a bacterial endotoxin test (BET) sample preparationmethod that overcomes the low endotoxin recovery (LER) effect that isdue to endotoxin masking.

Protein therapeutics (such as monoclonal antibodies) are often generatedusing genetically transformed eukaryotic and prokaryotic cells, such ase.g. bacteria. Used for bacterial productions are fast growing bacteriasuch as Escherichia coli. However, during growth and cultivation of therecombinant protein highly toxic lipopolysaccharides (LPS) are secretedinto the medium. These components are denoted as bacterial endotoxins(short endotoxins). Gram-negative bacteria possess LPS as an essentialcomponent of their cell wall. A Gram-negative bacteria cell containsapproximately 3.5×10⁵ LPS molecules, which occupy an overall area ofapproximately 4.9 μm² (Rietschel, 1994, FASEB J. 8:217-225). In the caseof E. coli, it means that LPS represent about three-quarters of thetotal bacterial cell surface. Approximately 10,000 CFU (colony formingunits) of a Gram-negative bacterium species correspond to 1 EndotoxinUnit (EU) (Rietschel, 1994, FASEB J. 8:217-225). EU refers toendotoxin/LPS; 1 EU≈100 pg LPS, depending on the LPS used. However, evenif products are not produced by recombinant means, most employedreagents are contaminated with endotoxin, as their production is rarelydone under aseptic or even sterile conditions. Therefore, LPS areubiquitous potential contaminants in case sterile and/or asepticconditions, during production of pharmaceuticals, cannot be kept. Amongall known bacterial compounds, endotoxin is one of the most toxicnatural compounds for mammals. LPS as present in the cell wall ofGram-negative bacteria are known to cause profound immunoactivationincluding the induction of fever when entering the human bloodstream. Itcauses delirious effects at extreme low concentrations (picogram-range)when entering the cardiovascular and lymphatic system, respectively.Unfortunately bacterial endotoxins are heat stable and their toxicity isnot linked to the presence of the bacterial cell at all. It is alsogenerally known that all protein therapeutics, irrespective of themethod of their production, must be expected or considered to becontaminated with low traces of bacterial endotoxins (so called “naturaloccurring endotoxin”, NOE). Therefore, endotoxin contamination remains acontinuous challenge for the production of pharmaceuticals such astherapeutic monoclonal antibodies. This has been outlined with emphasisvery clearly in the “Guidance for Industry, pyrogen and endotoxintesting”, issued by the Food and Drug Administration (FDA) in June 2012.

To ensure that injectable protein therapeutics (such as monoclonalantibodies) are safe for human use, endotoxin testing has to be done.Endotoxin testing is commonly performed using the compendial methods ofUS Pharmacopeia <85>, European Pharmacopeia 2.6.14 or JapanesePharmacopeia 4.01 with gel-clot, chromogenic or turbidimetric Limulusamoebocyte lysate (LAL) techniques (also designated as LAL assay or LALtest). The compendial name for the LAL assay is bacterial endotoxin(s)test (BET). The BET is used for detecting the presence of unsafe levelsof endotoxin, in particular of Gram-negative bacterial endotoxin, in agiven sample or substance.

The LAL assay is routinely performed with a diluted test sample alongwith a positive control, which is a sample with a known amount of spikedcontrol standard endotoxin (CSE). CSE is a defined form of endotoxincommercially available (supplied, e.g., by Lonza, Associates of CapeCod, Inc. (ACC), or Charles River Laboratories International, Inc.).According to the compendial LAL assay method qualification, CSE isspiked to a diluted sample at a non-interfering concentration (NIC) toachieve an acceptable recovery rate of 50-200%. This approach fails torecognize that components of the sample matrix of pharmaceuticalformulations as well as storage conditions potentially impact the LALreactivity of endotoxins present in undiluted product samples. Whenundiluted product samples are spiked with endotoxins such as CSEfollowed by LAL assay, low endotoxin recovery (<50%) was observed forcertain biologic products. Such low endotoxin recovery was particularlyobserved if the formulation of the product contained amphiphiliccompounds such as detergents. Detergents are added to the product inorder to solubilize the therapeutic protein. This masking of endotoxinresults in the significantly reduced detection of endotoxin, especiallyin case the LPS contamination is low. This phenomenon is called“endotoxin masking” if endotoxin recovery cannot be increased by sampledilution after spiking.

Different sample pre-treatments for the LAL assay to overcome assayinhibition and/or enhancement are known. However, at present thesesample pre-treatments do not lead to satisfactory results. Therefore,there is still a risk that endotoxin contaminations occur duringmanufacturing of pharmaceuticals that cannot be detected by the LALassay due to endotoxin masking. Based on current knowledge there are twodifferent types of endotoxin masking:

-   1) Endotoxin masking caused by endotoxin-binding proteins present in    the sample (“protein masking”, Petsch, Anal. Biochem. 259, 1998,    42-47). For example the formation of protein-endotoxin aggregates,    e.g. with human lipoproteins Apo A1, lysozyme, ribonuclease A or    human IgG, is well known to reduce LAL reactivity of endotoxins    (Emancipator, 1992; Petsch, Anal. Biochem. 259, 1998, 42-47).-   2) Endotoxin masking caused by certain formulation ingredients or    buffer components often present in pharmaceutical products. For    example, endotoxin masking specifically caused by a combination of    polysorbate plus either citrate or phosphate is termed “Low    Endotoxin Recovery” or LER (Chen, J. and Williams, K. L., PDA Letter    10, 2013, 14-16, Williams, American Pharmaceutical Review, Oct. 28,    2013: Endotoxin Test Concerns of Biologics). Endotoxin masking may    also be caused by any other buffer component and non-ionic detergent    or combinations thereof.

Due to the LER effect, potential endotoxin contaminations occurringduring manufacturing remain underestimated or undetected when aconventional LAL assay is used. The LER effect represents a continuouschallenge for pharmaceutical products (Hughes, BioPharm. AsiaMarch/April 2015, 14-25).

Accordingly, the technical problem underlying the present invention isthe provision of means and methods for overcoming the LER effect.

The technical problem has been overcome by the methods of the presentinvention as detailed below.

Herein is reported an improved LAL assay for quantification ofendotoxin. This improved LAL assay is particularly useful whenamphiphilic matrices mask endotoxin determination(Low-Endotoxin-Recovery; LER).

In particular, in context of the present invention it was surprisinglyfound that by the sequence of adding magnesium ions, e.g. in form ofMgCl₂, to a sample; diluting the sample; and dialyzing the sample havinga pH-value of 5.7-8.0, the LER effect can successfully be overcome. Or,in other words, the sample preparation method as reported herein issuitable for overcoming the LER effect in a LAL assay.

More specifically, in context of the present invention, a samplepreparation method for samples comprising an antibody (e.g. a sample ofa therapeutic monoclonal antibody) has been found. This inventive samplepreparation method has the advantage that it surprisingly andunexpectedly obviates the LER effect if a LAL assay is performed. Morespecifically, the present invention relates to a method for thepreparation of a sample comprising an antibody for BET (preferably for aLAL assay), wherein the method comprises the following steps in thefollowing order:

-   -   (a) adding magnesium ions, preferably in form of MgCl₂, to the        sample,    -   (b) diluting the sample, and    -   (c) dialyzing the sample having a pH-value of 5.7-9.0,        preferably of 5.7-8.0, against an endotoxin-free aqueous        solution.

Thus, according to the present invention, a sample comprising anantibody (e.g. a sample of a therapeutic monoclonal antibody) isprocessed by performing the steps (a) to (c) of the inventive samplepreparation method. These steps and their combination surprisingly leadto the provision of a sample, which does not suffer from the LER effectif a LAL assay is performed. Or, in other words, after performing thesteps (a) to (c) of the herein provided sample preparation method, thesample comprising an antibody is reactive to factor C in the LALenzymatic cascade. Thus, the inventive sample preparation method isadvantageously performed before determining bacterial endotoxin via theLAL assay. Accordingly, the present invention also relates to a methodfor determining (i.e. detecting and/or quantifying) endotoxin in asample. In particular, the herein provided endotoxin determinationmethod allows the determination (i.e. the detection and/orquantification) of endotoxin in a sample comprising an antibody (e.g. atherapeutic monoclonal antibody). In particular, the present inventionrelates to a method for determining bacterial endotoxin in a sample(that preferably exhibits a LER effect) comprising an antibody, whereinthe method comprises the following steps in the following order:

-   -   (a) adding magnesium ions, preferably in form of MgCl₂, to the        sample (i.e. to the sample comprising an antibody),    -   (b) diluting the sample,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0 against an        endotoxin-free aqueous solution, and    -   (d) determining bacterial endotoxin in the sample by using a LAL        assay.

Preferably, in the sample preparation method or the endotoxindetermination method of the present invention, 1.5-5 ml clear glass,crimp neck, flat bottom vessels are used. Most preferably, the vesselsare screw neck glass vials of Macherey-Nagel GmbH (1.5 ml or 4 ml).

Endotoxin contamination represents a high risk in the production ofpharmaceuticals such as monoclonal antibodies. In the prior artendotoxin testing, in particular for therapeutic antibodies, isperformed by using a conventional LAL assay. However, as demonstrated inthe appended Examples, the LAL assay fails to detect/underestimatesendotoxin contamination in antibody formulations that exhibit the LEReffect. Undetected/underestimated endotoxin represents an extreme safetyrisk for any pharmaceutical sample, particularly for pharmaceuticalsthat are administered intramuscularly or intravenously. However, despiteof its tremendous practical importance, nothing is known about thephysico-chemical mechanisms of the LER effect. Hence, the prior artfails to provide methods for the correct determination of endotoxin intherapeutic products that exhibit the LER effect.

In context of the present invention a robust physico-chemical set-up,which obviates the LER effect and results in satisfactory recovery ratesfrom CSE-spiked samples has been found. In particular, as demonstratedin the illustrative appended Examples, the methods as reported hereinallow the recovery of the CSE spiked to a given sample at a definedconcentration (0.5 or 5.0 EU/ml). Importantly, the herein providedmethods lead to recovery rates ranging between 50% and 200%, this wayfulfilling the requirements of the FDA. Thus, the present inventionadvantageously provides methods, which are able to unmask endotoxins andto overcome the LER effect. More specifically, in context of the presentinvention it has surprisingly been found that the specific combinationand sequence of the steps (a) to (c) (i.e. (a) adding magnesium ions tothe sample to be tested; (b) diluting the sample to be tested; and (c)dialyzing the sample to be tested (wherein the sample has a pH-value of5.7-8.0), obviates the LER effect of the sample to be tested forendotoxin. Or, in other words, performing the steps (a) to (c) unmasksthe endotoxin in the sample, and thus, makes the endotoxin detectablewith the LAL assay. The appended Examples show that the herein providedmethods overcome the LER effect e.g. in formulated rituximab. Bycontrast, the same protocol could not reveal satisfactory results forNeoRecormon® (which does not comprise an antibody but epoetin-beta).This indicates that the herein provided methods are particularly usefulfor obviating the LER effect in antibody formulations, preferably informulations with a monoclonal antibody, citrate buffer and polysorbate80.

Thus, the herein provided sample preparation method and the hereinprovided endotoxin determination method advantageously obviate the LEReffect. Therefore these methods improve the detection of endotoxin inpharmaceuticals. This leads to the production of pharmaceutical productswith less adverse effects. Consequently, the herein provided methodswill improve the state of health of the consumer and may save the livesof critically ill patients.

In the herein provided methods, the antibody that is comprised in thesample may have been produced in and/or purified from bacterial oreukaryotic cells. For example, the antibody may have been produced andpurified from Chinese hamster ovary (CHO) cells. In one aspect of theinvention, the sample (i.e. the sample comprising an antibody) is adissolved solid sample. In another aspect of the invention, the sample(i.e. the sample comprising an antibody) is a liquid sample. In theherein provided sample preparation method and endotoxin determinationmethod, it is envisaged that the antibody (i.e. the antibody that iscomprised in the sample) is a therapeutic antibody. Preferably, theantibody (i.e. the antibody that is comprised in the sample) is amonoclonal antibody. However, in the herein provided methods theantibody (that is comprised in the sample) may also be a polyclonalantibody. Herein, also multispecific antibodies (e.g., bispecificantibodies), or antibody fragments are comprised by the term “antibody”,so long as they exhibit the desired biological activity. The antibodymay be human, humanized, or camelized.

The herein provided methods advantageously render LER-prone samples of apharmaceutical formulation reactive to factor C in the LAL enzymaticcascade. The LER effect has been reported in biologic products, whichare formulated with amphiphilic compounds such as non-ionic detergents,in particular if they are combined with citrate or phosphate as buffer.The appended Examples demonstrate that the herein provided methodsreliably obviate the LER effect in such therapeutic formulations.Therefore, it is envisaged in context of the herein provided samplepreparation method and endotoxin determination method that saidtherapeutic antibody (i.e. the therapeutic antibody that is comprised inthe sample) is formulated with at least one detergent (preferably apolysorbate).

However, it is envisaged that said therapeutic antibody is formulatedwith a polysorbate that does not comprise a structural motif for thelipid A cavity in the C reactive protein of the LAL cascade. Morespecifically, straight chain fatty acids such as lauric acid may mimicthe fatty acids in the lipid A molecule of the LAL cascade, as thismolecule also contains fatty acids with 12 carbon atoms and no doublebonds (i.e. C:D is 12:0). Such straight fatty acids may negativelyinterfere with the LAL cascade. Therefore, in the herein providedmethods, it is envisaged that said therapeutic antibody is notformulated with a detergent that comprises straight fatty acids such aslauric acid. Polysorbate 20 comprises lauric acid. Thus, it is envisagedin the herein provided methods that the sample (in particular the sampleof a therapeutic antibody) is not formulated with polysorbate 20. Alsophosphate buffer, particularly sodium phosphate buffer, may interferewith the LAL cascade. Therefore, these buffers are less useful for theherein provided sample preparation methods. Accordingly, the inventionrelates to the herein provided sample preparation method or endotoxindetermination method, wherein the (therapeutic) antibody that iscomprised in the sample is not diluted with a phosphate buffer. In oneaspect of the present invention the sample does not comprise more than0.1 mM phosphate buffer and does not comprise a concentration ofpolysorbate 20 that is higher than 1/100 of its critical micellarconcentration (CMC). In a preferred aspect of the present invention, thesample does either not comprise phosphate buffer and polysorbate 20, orcomprises an amount of phosphate buffer and/or polysorbate 20 that isblow the detection limit when using standard detection methods.

As demonstrated in the appended Examples by using the methods of theinvention, the LER effect can be overcome in formulated rituximabsamples as well as in rituximab placebo samples. Rituximab placebosamples only differ from rituximab samples in that the antibody isabsent. Beside this difference, rituximab placebo samples contain allthe other components of the formulation of rituximab such as detergentand buffer. This indicates that the herein provided methods do notdepend on a formulation comprising a particular monoclonal antibody butcan be used, e.g., to obviate the LER effect in every formulationexhibiting this effect. Such formulations include formulationscomprising polysorbate 80 and a chelating buffer (such as sodiumcitrate). This formulation is typical for antibodies, in particularmonoclonal antibodies. Thus, the above described method is expected tobe useful to overcome the LER effect in every monoclonal antibodyformulation. Rituximab is formulated with a mixture of polysorbate 80and sodium citrate buffer (i.e. 25 mM sodium citrate buffer, pH 6.5; 700mg/l polysorbate 80, and 154 mM NaCl). It is envisaged in context of thepresent invention, that the sample comprising an antibody has thisformulation.

The appended Examples demonstrate that in exemplary samples oftherapeutic antibodies that are formulated with polysorbate 80 andcitrate buffer, the LER effect can be overcome by using the hereinprovided methods. Therefore, in the herein provided sample preparationmethod or endotoxin determination method it is preferred that the sample(i.e. the sample comprising an antibody) is formulated with polysorbate80. Accordingly, in the herein provided methods, it is envisaged thatthe sample (i.e. the sample comprising an antibody) comprisespolysorbate 80. Preferably, the sample comprises 500-1000 mg/lpolysorbate 80, more preferably about 700 mg/l polysorbate 80. It isfurther envisaged in the herein provided methods that the sample (i.e.the sample comprising an antibody) is formulated with a chelating buffer(such as citrate buffer). Said citrate buffer may be a 5-50 mM citratebuffer, pH 6.0-7.0; preferably a 25 mM citrate buffer, pH 6.5.Preferably, the citrate buffer is a sodium citrate butter. For example,in the herein provided methods, the sample (i.e. the sample comprisingan antibody) may comprise 5-50 mM Na-citrate, preferably 25 mMNa-citrate. Most preferably, the sample comprises polysorbate 80 andsodium citrate buffer. For example, the sample may comprise about 700mg/l polysorbate 80 and 5-50 mM, preferably about 25 mM sodium citratebuffer. Most preferably, in the herein provided methods the sample is asample of an antibody, which is formulated with an about 25 mMNa-citrate buffer and about 700 mg/l polysorbate 80 and has a pH valueof about 6.5.

In the herein provided sample preparation method or endotoxindetermination method it is preferred that said antibody (i.e. theantibody that is comprised in the sample) is an anti-CD20 antibody. Morepreferably, the antibody is the anti-CD20 antibody rituximab. The aminoacid sequences of the heavy and light chain of rituximab are shownherein as SEQ ID NOs: 1 and 2, respectively. The person skilled in theart readily knows how to obtain a coding nucleic acid sequence from agiven amino acid sequence. Thus, with the knowledge of SEQ ID NOs: 1 and2, a coding nucleic acid sequence of rituximab can easily be obtained.Rituximab is commercially available, e.g., as Rituxan® and MabThera®, orZytux®.

In step (a) of the herein provided sample preparation method orendotoxin determination method, magnesium ions (Mg²⁺), e.g. in form ofMgCl₂, are added to the sample (i.e. to the sample comprising anantibody). Herein, the term “magnesium chloride” or “MgCl₂” refers tothe chemical compounds with the formula MgCl₂ as well as its varioushydrates MgCl₂ (H₂O)_(x) (i.e. MgCl₂.x H₂O). For example, in step (a) ofthe herein provided methods MgCl₂ hexahydrate (i.e. MgCl₂.6H₂O) may beadded to the sample. The illustrative appended Examples demonstrate thatin step (a) addition of magnesium ions to a final concentration of10-100 mM Mg²⁺ markedly reduces the LER effect. Moreover, the appendedExamples also show that a concentration of Mg²⁺ that is twice theconcentration of the buffer of the sample results in best endotoxinrecovery rates. For example, when rituximab was used as a sample, bestendotoxin recovery rates were obtained when in step (a) the addition ofthe magnesium salt MgCl₂ results in a final concentration of Mg²⁺ thatis twice of the sodium citrate concentration (i.e. 50 mM Mg²⁺).Therefore, in step (a) of the herein provided methods it is envisagedthat magnesium ions in form of a salt (e.g. MgCl₂) are added to resultin a final Mg²⁺ concentration that is twice the concentration of thebuffer (e.g. the sodium citrate buffer). For example, in the methodsprovided herein, preferably magnesium ions are added to the sample sothat the final concentration of Mg²⁺ [in step (a)] is 10-100 mM Mg²⁺,more preferably 25-75 mM Mg²⁺, even more preferably 40-75 mM Mg²⁺, andmost preferably about 50 mM Mg²⁺ (i.e. 45-55 mM Mg²⁺). Or, if the samplealready comprises magnesium ions, then the added amount of Mg²⁺ isadjusted so that the resulting final concentration of Mg²⁺ [in step (a)]is preferably 10-100 mM, more preferably 25-75 mM, even more preferably40-75 mM, and most preferably of about 50 mM Mg²⁺ (i.e. 45-55 mM Mg²⁺).After step (a), i.e. in step (b), the sample is diluted. However, instep (a), the term “adding magnesium ions to a concentration of . . . ”or grammatical variations thereof and the term “adding magnesium ions toa final concentration of . . . ” or grammatical variations thereof,refer to the final concentration of Mg²⁺ in step (a). For example,adding in step (a) MgCl₂ to a (final) concentration of 45-55 mM MgCl₂means that after addition of MgCl₂ in step (a) the concentration ofMgCl₂ is 45-55 mM. Accordingly, if, e.g., in step (b) the sample isdiluted at a ratio of 1:10 (sample:buffer/water), the concentration ofthe magnesium ions and likewise that of MgCl₂ is 4.5-5.5 mM.

The appended Examples demonstrate that an incubation step after additionof magnesium ions further improves the recovery rates in the LAL assay.Therefore, in the herein provided methods, after addition of magnesiumions the sample is preferably incubated for 30 min to 6 hours, morepreferably for 1-4 hours, most preferably for about 1 hour. In oneprioritized aspect of step (a) of the herein provided methods the sampleis incubated for about 1 hour at room temperature after addition of themagnesium ions. Before and after said incubation step, the sample may beshaked [e.g. in the Heidolph Multi Reax shaker, high speed (2,037 rpm)].For example, before and after the incubation step the sample may beshaked for 30 sec to 10 min, preferably for 1 min.

In step (b) of the herein provided sample preparation method orendotoxin determination method, the sample (i.e. the sample comprisingan antibody) is diluted. The sample may be diluted with endotoxin-freewater. The appended Examples demonstrate that good recovery rates can beobtained if during dialysis the sample has a pH-value of 5.7-8.0. Evenbetter recovery rates were obtained if during dialysis the sample had apH-value of 6.0-8.0. Best recovery rates were obtained if duringdialysis the sample had a pH-value of 6.5-7.5. Thus, one aspect of theinvention relates to the herein provided methods, wherein in step (b)the sample is diluted with endotoxin-free water, and wherein afterdilution and prior to dialysis the pH-value of the sample is adjusted to5.7-8.0, more preferably to 6.0-8.0, most preferably to 6.5-7.5. Thus,in one aspect of the invention, in step (b) of the herein providedmethods the pH-value of the sample is adjusted to pH 5.7-8.0, morepreferably to pH 6.0-8.0. Most preferably, in step (b) of the hereinprovided methods the pH-value of the sample is adjusted to pH 6.5-7.5.For example, the pH-value of the sample may be adjusted to pH 5.7, pH5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH6.7, pH 6.8, pH 6.9, or pH 7.0. However, it is preferred herein that thepH-value of the sample is adjusted in step (b) by diluting the samplewith 10-50 mM buffer, e.g. Tris/HCl-buffer, pH 6.0-9.0, more preferablywith 10-50 mM buffer, e.g. Tris/HCl buffer, pH 6.0-8.0. Therefore, it isenvisaged in a preferred aspect of the herein provided methods that instep (c) the pH-value of the sample is adjusted by diluting the samplewith 10-50 mM Tris/HCl buffer, pH 6.0-9.0. More preferably, the pH-valueof the sample is adjusted by diluting the sample with a 10-50 mMTris/HCl buffer, pH 6.0-8.0. Most preferably, the pH-value of the sampleis adjusted by diluting the sample (in step (b)) with 50 mM Tris/HCl pH˜7.0. Thus, in the herein provided methods, during dialysis in step (c)the sample has a pH-value of 5.7-8.0, preferably of 6.0-8.0, morepreferably 6.5-7.5.

As indicated above, the sample can comprises a detergent such aspolysorbate 80. The appended illustrative Examples demonstrate thatinter alia dilution of a sample comprising a detergent (e.g. polysorbate80) renders the endotoxin molecules accessible in the LAL assay. Withoutbeing bound by theory it is believed that dilution of a samplecomprising a detergent to near-CMC concentrations reduces the micellarcompartmentalization of the sample, and therefore reduces the LEReffect.

The appended Examples show that a dilution of 1:5 to 1:20 considerablyinfluences the recovery rate in a LAL assay. Thus, the invention relatesto the herein provided sample preparation method and endotoxindetermination method, wherein in step (b) the sample is diluted at aratio of 1:5 to 1:20 (sample:buffer/water), preferably of 1:10(sample:buffer/water). In the herein provided methods the antibody ispreferably formulated with an about 25 mM sodium citrate buffer andabout 700 mg/l polysorbate 80. Thus, in the herein provided methods, thesample may be diluted in step (b) such that the concentration of thebuffer decreases to 5-1.25 mM, preferably to 2.5 mM. In addition, thesample may be diluted in step (b) such that the concentration of thedetergent decreases to 140-35 mg/l, preferably to 70 mg/l. In theappended Examples, the samples were antibody formulations having aconcentration of the antibody of about 10 mg/ml. These samples werediluted in step (b) of the inventive methods to result in an antibodyconcentration of 2-0.5 mg/ml. Thus, in the herein provided methods, thesample may be diluted in step (b) such that the concentration of theantibody decreases to 2-0.5 mg/ml, preferably to 1 mg/ml. In the hereinprovided methods also an undiluted control may be prepared. Saidundiluted control is treated in the same way as the sample to be tested,with the exception that the undiluted control is not diluted (in step(b)). Herein, “buffer/water” means “buffer or water”.

In step (c) of the herein provided sample preparation method andendotoxin determination method, the sample is dialyzed against anendotoxin-free aqueous solution. The endotoxin-free aqueous solution maybe endotoxin-free water. However, said endotoxin-free aqueous solutionmay also be an endotoxin-free aqueous solution that comprises magnesiumions, e.g. added in form of the salt MgCl₂. Accordingly, one aspect ofthe invention relates to the herein provided methods, wherein in step(c) the endotoxin-free aqueous solution contains magnesium ions, e.g.2.5-10 mM MgCl₂.

Before starting the dialysis, the samples may be shaked [e.g. in aHeidolph Multi Reax shaker, high speed (2,037 rpm) at room temperature],e.g. for 30 sec to 10 min, preferably for 1 min. Preferably, thedialysis in step (c) is for 1-48 hours, more preferably for 4-24 hours,most preferably for about 24 hours. Thus, it is preferred that in step(c) the dialysis is for about 24 hours. The dialysis may be performed at15-30° C., preferably at room temperature (i.e. 21±2° C.). Afterdialysis, the sample may be shaked [e.g. in a Heidolph Multi Reaxshaker, high speed (2,037 rpm) at room temperature, e.g. for 20 min to 1hour, preferably for (at least) 20 min.

The dialysis may be performed by using a Spin Dialyzer (e.g. the HarvardSpinDIALYZER, catalogue Nb. 74-0314) or a Fast Spin Dialyzer (e.g. theHarvard Fast Spin Dialyzer, catalogue Nb. 74-0412). It is preferred(especially if a Fast Spin Dialyzer is used) that the rotation frequencyof the stirrer is high, meaning that the frequency of the stirrer is 50to 300 rpm, preferably 200 to 300 rpm. The stirrer has preferably alength of 20-60 mm and a diameter (i.e. cross-section dimension) of 5-25mm. More preferably, the stirrer has a length of about 40 mm and adiameter of about 14 mm. The stirrer is most preferably aheat-sterilized (e.g. 4 hours at 250° C.) magnetic stirrer having alength of about 40 mm and a diameter of about 14 mm. Such stirrers areavailable from OMNILAB. Indeed dialysis is usually done with a highfrequency of the stirrer, as this facilitates diffusion through thedialysis membrane. Accordingly, dialyzing with a high frequency of thestirrer is the standard dialysis procedure. The vessel that is used fordialysis has preferably a volume of 500-5000 ml, more preferably1000-3000 ml, most preferably 1500-2500 ml. This vessel may have adiameter of 120 mm and a height of 240 mm. For example, the vessel thatis used for dialysis may be a DURAN® beaker, tall form, 2000 ml (e.g.available from OMNILAB, Germany, P/N: 5013163). Using a Fast SpinDialyzer is preferred as it has the double area of dialysis membrane,and thus is believed to be suitable for a more efficient and quickerdialysis.

It is envisaged that for the dialysis in step (c) a membrane with amolecular-weight cut-off of 100 Da to 16 kDa, preferably of 500 Da to 10kDa, most preferably of 10 kDa is used. For the dialysis in step (c) acellulose ester or a cellulose acetate membrane may be used. Preferably,for the dialysis in step (c) a cellulose acetate membrane is used. Mostpreferably, a cellulose acetate membrane with a molecular-weight-cut-offof 10 kDa is used during the dialysis.

Thus, in step (c) of the herein provided methods, the dialysis ispreferably performed for about 24 hours by using a cellulose acetatemembrane with a molecular-weight cut-off of 10 kDa. Before the dialysis,the dialysis membrane may be washed, preferably in endotoxin-free water.In particular, the dialysis membrane may be shaked (e.g. with the ShakerSG 20. IDL GmbH, Germany or equivalent, 50 to 300 rpm, preferably 100rpm) in endotoxin-free water. For example, the dialysis membrane may bewashed by shaking it for 10 min to 3 hours, preferably for 1 hour inendotoxin-free water. After this washing step, the dialysis membrane ispreferably transferred in fresh endotoxin-free water and again washed byshaking it for 10 in to 3 hours, preferably for 1 hour.

The dialysis may be performed in 1 ml chambers, e.g. in 1 ml spindialyzer (Harvard) chambers equipped with a membrane (such as celluloseacetate membrane) having a molecular-weight cut-off ranging from 500 Dato 10 kDa (e.g. a molecular-weight cut-off of 10 kDa). During dialysisthe water is preferably changed, more preferably the water is changedtwice. For example, the water may be changed after 2 and 20 hours ofdialysis or after 18 and 22 hours of dialysis. Preferably, the water ischanged after 2 and 4 hours of dialysis.

It is preferred in context of the herein provided methods that after thedialysis in step (c) the sample is shaked [e.g. in a Heidolph Multi Reaxshaker, high speed (2,037 rpm) at room temperature. Preferably, thesample (i.e. the sample comprising an antibody) is shaked after dialysisfor 10 min to 1 hour, more preferably for 20 min. In addition oralternatively to shaking, the sample may be treated with ultrasoundafter dialysis. Thus, one aspect of the invention relates to the hereinprovided methods, wherein in step (c) the sample is treated withultrasound after dialysis.

If the herein provided sample preparation method is combined with a LALassay, then this combined method advantageously reaches the FDArequirements for the quantitative and reproductive detection of adefined amount of CSE spiked to a sample. Preferably, the LAL assay ofthe herein provided methods is a LAL assay as described below.

The appended Examples indicate that addition of Mg²⁺ (i.e. magnesiumions) has the further advantage that it retains the endotoxin in theinner compartment of the dialysis chamber. Thus, the dialysis in step(c) leads only to the removal of the buffer (e.g. the sodium citratebuffer) and not of the endotoxin. The dilution step may reduce theconcentration of the detergent (e.g. polysorbate 80) so as to abolishthe inhibition of the LAL cascade by the detergent. The appendedExamples demonstrate that the LER effect can in particular reproduciblybe overcome if the steps of the inventive methods are performed in theorder: (1) addition of Mg²⁺; (2) dilution; and (3) dialysis.Accordingly, the combination of the steps (1), (2) and (3), or thecombination of the claimed steps (a), (b) and (c) reproducibly overcomesthe LER effect. The preferred amount of Mg²⁺ that is to be added, thepreferred degree of dilution and the preferred parameters for dialysisare detailed herein above and below.

In a preferred aspect, the invention relates to the herein providedmethod for the preparation of a sample comprising an antibody for a LALassay, wherein the method comprises the following steps in the followingorder:

-   -   (a) adding magnesium ions, e.g. in form of MgCl₂, to the sample        to a final concentration of 10-100 mM, preferably 40-75 mM, most        preferably 45-55 mM,    -   (b) diluting the sample at a ratio of 1:5 (sample:buffer) to        1:20 (sample:buffer), preferably 1:10 (sample:buffer) with 10-50        mM Tris/HCl buffer, pH 6.0-8.0; preferably with 50 mM        Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) against endotoxin-free water for 1-48        hours, preferably for 4-24 hours, most preferably for 24 hours.        Preferably a cellulose acetate membrane with a molecular weight        cut-off of 10 kDa is used and the water is changed after 2 and 4        hours. Most preferably, a Fast Spin Dialyzer is used and the        frequency of the stirrer is 50 to 300 rpm preferably 200 rpm.

As mentioned above, the antibody is preferably a monoclonal antibody.More preferably, the antibody is rituximab. Most preferably, in theherein provided methods the sample is a sample of an antibody, which isformulated with an about 25 mM sodium citrate buffer (7.35 mg/ml) andabout 700 mg/l polysorbate 80 and has a pH value of about 6.

The term “about” and the symbol “˜” are used interchangeably herein andspecify that the specific value provided may vary to a certain extent.For example, “about” or “˜” (e.g. in the context of about/˜25 mM sodiumcitrate buffer) means that variations in the range of ±10%, preferably±5%, most preferably ±2% are included in the given value.

As indicated, it is envisaged in context of the present invention thatthe herein provided sample preparation method is combined with a LALassay. The LAL assay has the advantage that it detects endotoxin at lowconcentration.

As given by the CSE standard curve the validated lower limit ofendotoxin detection is 0.005 EU/mL in kinetic chromogenic LALtechniques. The LAL reagent of these techniques comprises the completeenzymatic amplification cascade of serine proteases purified from theLimulus crab.

The lower limit of endotoxin (CSE) detection in the more recentlydeveloped EndoLISA® assay (Hyglos GmbH, Germany) is indicated by themanufacturer to be 0.05 EU/mL (Advertisment of Hyglos: Grallert et al.in: Nature Methods, October 2011;p://www.hyglos.de/fileadmin/media/Application_note_EndoLISA_Nature_Methods_October_2011.pdf).This EndoLISA® assay employs a recombinant form of only the initialenzyme of the Limulus cascade, i.e. factor C. Distinct from certifiedLAL tests the EndoLISA® assay additionally includes an initial endotoxinadsorption step provided by a pre-coating of the micotiter plate by abacteriophage-encoded protein that yet has not been proven to bind thebroad spectrum of bacterial endotoxins well known to be detected by theLAL method.

In particular, in a preferred aspect the present invention relates to amethod for determining bacterial endotoxin in a sample comprising apolypeptide, wherein the method comprises the following steps in thefollowing order:

-   -   (a) adding magnesium ions, preferably in form of MgCl₂, to the        sample to a final concentration of 10-100 mM, preferably 40-75        mM, most preferably 45-55 mM,    -   (b) diluting the sample at a ratio of 1:5 (sample:buffer) to        1:20 (sample:buffer), preferably 1:10 (sample:buffer) with 10-50        mM Tris/HCl buffer, pH 6.0-8.0; preferably with 50 mM        Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) against endotoxin-free water for 1-48        hours, preferably for 4-24 hours, most preferably for 24 hours.        Preferably a cellulose acetate membrane with a molecular weight        cut-off of 10 kDa is used and the water is changed after 2 and 4        hours. Most preferably, a Fast Spin Dialyzer is used and the        frequency of the stirrer is 50 to 300 rpm preferably 200 rpm.    -   (d) Determining bacterial endotoxin in the sample by using a LAL        assay.

As mentioned above, the antibody is preferably a monoclonal antibody.More preferably, the antibody is rituximab. Most preferably, in theherein provided methods the sample is a sample of an antibody, which isformulated with an about 25 mM sodium citrate buffer and about 700 mg/lpolysorbate 80 and has a pH value of about 6.5.

As indicated in the appended Examples, a “LER positive control” (alsodesignated as “positive LER control”) may be used in the LAL assay ofstep (d) of the herein provided endotoxin determination method. Said“LER positive control” is an indicator to demonstrate that the sample tobe tested (i.e. the sample comprising an antibody) would exhibit the LEReffect if the steps (a) to (c) of the herein described methods would nothave been performed. Or, in other words, the “LER positive control” isused in a LAL assay as a positive control to show that a known spikedamount of endotoxin (within the sample to be tested) cannot be recoveredby using a LAL assay only (i.e. without performing steps (a) to (c) ofthe herein provided methods). In context of the present invention it hassurprisingly and unexpectedly been found that a positive LER effect canonly be obtained if, after spiking the sample with CSE, the sample isshaked for 45 min to 2 hours, preferably for about 60 min to 2 hours,most preferably for about 60 min.

Thus, in context of the present invention the “LER positive control” isprepared by spiking a known amount of endotoxin into an aliquot of thesample to be tested for endotoxin (e.g. into an aliquot of the samplecomprising an antibody) and shaking the spiked sample for 45 min to 2hours, preferably for about 60 min to 2 hours, most preferably for about60 min. Thus, the invention relates to the herein provided endotoxindetermination method, further comprising producing a LER positivecontrol by spiking a known amount of endotoxin into an aliquot of thesample and shaking the endotoxin spiked aliquot of the sample for ≥60min (more preferably for 60 min to 2 hours).

Preferably, in the herein provided method for determining bacterialendotoxin the “LER positive control” is prepared by spiking CSE to afinal concentration of 5.0 EU/ml to an aliquot of the sample to betested. Afterwards, the spiked aliquot is shaked [e.g. in a HeidolphMulti Reax shaker, high speed (2,037 rpm) at room temperature] for ≥60min, most preferably for 60 min. After shaking, the endotoxin spikedaliquot is preferably diluted to the same extend as the sample to betested in step (b) of the herein provided methods. Preferably, thespiked aliquot is diluted with endotoxin-free water. After dilution, thespiked aliquot is preferably shaked [e.g. in a Heidolph Multi Reaxshaker, high speed (2,037 rpm) at room temperature], e.g. for 1 min.

Thus, the “LER positive control” is preferably prepared by the followingprocedure in the following order:

-   -   Spiking CSE to a final concentration of 5.0 EU/ml to an aliquot        of the sample to be tested. Preferably, the “LER positive        control” is prepared in a 1.5-5 ml clear glass, crimp neck, flat        bottom vessel, more preferably in a screw neck glass vial of        Macherey-Nagel GmbH (1.5 ml or 4 ml)    -   Shaking the spiked aliquot for ≥60 min (more preferably for 60        min to 2 hours), most preferably for 60 min. Preferably, the        spiked aliquot is shaked at high speed (2,037 rpm) at room        temperature (i.e. 21±2° C.). Most preferably the spiked aliquot        is shaked at high speed (2,037 rpm) in a Heidolph Multi Reax        shaker at room temperature.    -   Diluting the spiked aliquot with endotoxin-free water. The        spiked aliquot is diluted to the same extend as the sample to be        tested in step (b) of the herein provided methods (i.e. if the        sample to be tested is diluted in step (b) at a ratio of 1:10,        then also the spiked aliquot is diluted at a ratio of 1:10).    -   Shaking the spiked aliquot (e.g. for 1 min).

As described above, during preparation of the “LER positive control”, adilution is performed. However, it is envisaged in context of thepresent invention that, beside said dilution, the “LER positive control”is not treated as described in steps (a) to (c) of the herein providedmethods. However, said “LER positive control” is used in step (d) of themethod for determining bacterial endotoxin to show that the sample to betested (i.e. the sample comprising an antibody) would exhibit the LEReffect if the steps (a) to (c) of the herein described methods would notbe performed. The “LER positive control” may be prepared during the timeof any one of steps (a) to (c) (e.g. during dialysis-time) so that it isready for use when the LAL assay is performed.

To identify that a given material (e.g. a buffer or a sample of atherapeutic antibody) exhibits the LER effect, endotoxin contents can bemonitored over time, e.g. in an endotoxin hold time study. Endotoxinhold time studies require endotoxin spiking of an undiluted sample andstorage of the endotoxin spiked sample over time. For example, thesample may be stored up to several months. Preferably, in a hold timestudy the endotoxin spiked sample is stored for several (e.g. 7 for upto 28) days and at defined time points a LAL assay is performed.Recovery rates that are lower than 50% of the amount of the spikedendotoxin indicate that the sample exhibits a LER effect.

As mentioned above, the LAL assay is routinely performed with a dilutedtest sample along with a diluted positive control (PPC), which is asample with a known amount of spiked CSE. Thus, in the LAL assay, whichis performed in step (d) of the herein provided endotoxin determinationmethod, it is envisaged that every sample is measured each time induplicate with a spiked control standard endotoxin (PPC) and withoutspiked endotoxin. Consequently, with every given sample, it can easilybe tested whether the herein provided sample preparation method or theherein provided endotoxin determination method has the favorable effectthat the endotoxin present in the sample (or at least 50-200% thereof asrequired by the FDA) can be detected by using the LAL assay. Thus, it isenvisaged that the LAL assay in step (d) of the herein providedendotoxin determination method comprises that a positive control (PPC)is tested along with the sample to be tested (i.e. the sample comprisingan antibody to be tested). Said positive control is identical to thesample to be tested with the exception that the PPC is spiked with aknown amount of CSE. Or, in other words, steps (a) to (c) of the hereinprovided methods have to be performed with the PPC in the same way aswith the sample to be tested. Accordingly, the PPC is prepared beforestep (a) of the herein provided methods.

In context of the present invention it has surprisingly and unexpectedlybeen found that a positive LER effect can only be obtained if, afterspiking the sample with CSE, the sample is shaked for 45 min to 2 hours,preferably for about 60 min to 2 hours, most preferably for about 60min. Thus, in context of the present invention the PPC is shaked [e.g.in a Heidolph Multi Reax shaker, high speed (2,037 rpm)] after spikingfor 45 min to 2 hours, preferably for about 60 min to 2 hours, mostpreferably for about 60 min. More preferably, the PPC is shaked [e.g. ina Heidolph Multi Reax shaker, high speed (2,037 rpm)] after spiking forabout 60 min at room temperature.

Thus, a preferred aspect of the invention relates to the herein providedmethod for determining bacterial endotoxin in a sample comprising anantibody, wherein the method comprises the following steps in thefollowing order:

-   -   (a0) preparing a PPC by        -   spiking a known amount of endotoxin into a first aliquot of            the sample comprising an antibody, and        -   shaking the endotoxin spiked aliquot for 60 min to 2 hours            (preferably for about 60 min at room temperature),    -   (a) adding magnesium ions to a second aliquot of the sample to        be tested as well as to the PPC,    -   (b) diluting the second aliquot of the sample to be tested as        well as the PPC,    -   (c) dialyzing the second aliquot of the sample having a pH-value        of 5.7-8.0 (preferably 5.8-7.0) to be tested as well as the PPC        against an endotoxin-free aqueous solution, wherein the sample        to be tested as well as the PPC have a pH-value of 5.7-9.0, and    -   (d) determining bacterial endotoxin in the second aliquot of the        sample to be tested as well as in the PPC by using a LAL assay.

In one aspect of the invention, the PPC is spiked with endotoxin suchthat a final endotoxin concentration of 5.0 EU/ml is obtained.

All the aspects and definitions disclosed in connection with the hereinprovided method for determining bacterial endotoxin apply, mutatismutandis, to said method if a PPC is applied. Thus, a preferred aspectof the invention relates to the herein provided method for determiningbacterial endotoxin in a sample comprising an antibody, wherein themethod comprises the following steps in the following order:

-   -   (a0) preparing a PPC by        -   spiking a known amount of endotoxin into a first aliquot of            the sample comprising an antibody, and        -   shaking the endotoxin spiked aliquot for ≥60 min (preferably            for 60 min at room temperature),    -   (a) adding magnesium ions, preferably in form of MgCl₂, to a        second aliquot of the sample to a final concentration of 10-100        mM, preferably 40-75 mM, most preferably 45-55 mM,    -   (b) diluting the second aliquot of the sample at a ratio of 1:5        (sample:buffer) to 1:20 (sample:buffer), preferably 1:10        (sample:buffer) with 10-50 mM Tris/HCl-buffer, pH 6.0-8.0,        preferably with 50 mM Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) against endotoxin-free water for 1-48        hours, preferably 4-24 hours, most preferably 24 hours.        Preferably a cellulose acetate membrane with a molecular weight        cut-off of 10 kDa is used and the water is changed after 2 and 4        hours. More preferably, a Fast Spin Dialyzer is used and the        frequency of the stirrer is high.    -   (d) Determining bacterial endotoxin in the sample by using a LAL        assay.

Additionally water controls can be applied in the herein providedendotoxin determination method. Preferably, at least two water controlsare used; wherein one consisting of endotoxin-free water and the otherof endotoxin-free water, which is spiked with a known amount ofendotoxin (e.g. resulting in a final concentration of 5.0 EU/ml CSE).The water controls are treated in the same manner as the sample to betested.

As indicated above, in the herein provided sample preparation method aswell as in the herein provided endotoxin determination method, it isenvisaged that in step (a), the sample is incubated for 30 min to 6hours, preferably for 1-4 hours, most preferably for 1. Moreover, it isalso envisaged that after dialysis the sample is shaked [e.g. in aHeidolph Multi Reax shaker, high speed (2,037 rpm) at room temperature],e.g. for 10 min to 1 hour, preferably for 20 min. Thus, one aspect ofthe invention relates to the herein provided method for the preparationof a sample comprising an antibody for a LAL assay, wherein the methodcomprises the following steps in the following order:

-   -   (a) adding magnesium ions, preferably in form of MgCl₂, to the        sample to a final concentration of 10-100 mM, preferably 40-75        mM, most preferably 45-55 mM; and incubating the sample for 30        min to 6 hours, preferably for 1-4 hours, most preferably for 1        hour,    -   (b) diluting the sample at a ratio of 1:5 (sample:buffer) to        1:20 (sample:buffer), preferably 1:10 (sample:buffer) with 10-50        mM Tris/HCl-buffer, pH 6.0-8.0, preferably with 50 mM        Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) against endotoxin-free water for 1-48        hours, preferably for 4-24 hours, most preferably for 24 hours.        (Preferably, a cellulose acetate membrane with a molecular        weight cut-off of 10 kDa is used and the water is changed after        2 and 4 hours. Most preferably, a Fast Spin Dialyzer is used and        the frequency of the stirrer is high.) After dialysis, the        samples are shaked for 10 min to 1 hour, preferably for 20 min.

Analogously, a further aspect of the invention relates to the hereinprovided method for determining bacterial endotoxin in a samplecomprising an antibody exhibiting a LER effect, wherein the methodcomprises the following steps in the following order:

-   -   (a) adding magnesium ions, preferably in form of MgCl₂, to the        sample to a final concentration of 10-100 mM, preferably 40-75        mM, most preferably 45-55 mM; and incubating the sample for 30        min to 6 hours, preferably for 1-4 hours, most preferably for 1        hour (wherein the sample may be shaked before and after the        incubation),    -   (b) diluting the sample at a ratio of 1:5 (sample:buffer) to        1:20 (sample:buffer), preferably 1:10 (sample:buffer) with 10-50        mM Tris/HCl-buffer, pH 6.0-8.0, preferably with 50 mM        Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) against endotoxin-free water for 1-48        hours, preferably for 4-24 hours, most preferably for 24 hours.        (Preferably, a cellulose acetate membrane with a molecular        weight cut-off of 10 kDa is used and the water is changed after        2 and 4 hours. Most preferably, a Fast Spin Dialyzer is used and        the frequency of the stirrer is high.) After dialysis, the        samples are shaked for 10 min to 1 hour, preferably for 20 min.    -   (d) Determining bacterial endotoxin in the sample by using a LAL        assay.

Moreover, as mentioned above, in herein provided endotoxin determinationmethod it is envisaged that a PPC is prepared and that the PPC is shakedfor 60 min to 2 hours after spiking. Thus, the present invention relatesto the herein provided method for determining bacterial endotoxin in asample comprising an antibody exhibiting a LER effect, wherein themethod comprises the following steps in the following order:

-   -   (a0) preparing a PPC by        -   spiking a known amount of endotoxin (e.g. to a final            concentration of 5.0 EU/ml) into a first aliquot of the            sample comprising an antibody, and        -   shaking the endotoxin spiked aliquot for 60 min to 2 hours            (preferably for 60 min at room temperature),    -   (a) adding magnesium ions, preferably in form of MgCl₂, to a        second aliquot of the sample as well as to the PPC to a final        concentration of 10-100 mM, preferably 40-75 mM, most preferably        45-55 MgCl₂; (and preferably incubating the sample and the PPC        for 30 min to 6 hours, more preferably for 1-4 hours, most        preferably for 1 hour, (wherein the sample may be shaked before        and after the incubation)),    -   (b) diluting the sample and the PPC at a ratio of 1:5        (sample/PPC:buffer) to 1:20 (sample/PPC:buffer), preferably 1:10        (sample/PPC:buffer) with 10-50 mM Tris/HCl-buffer, pH 6.0-8.0,        preferably with 50 mM Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) and the PPC against endotoxin-free water        for 1-48 hours, preferably for 4-24 hours, most preferably for        24 hours. (Preferably, a cellulose acetate membrane with a        molecular weight cut-off of 10 kDa is used and the water is        changed after 2 and 4 hours. Most preferably, a Fast Spin        Dialyzer is used and the frequency of the stirrer is high.)        After dialysis, the samples and the PPC are shaked for 10 min to        1 hour, preferably for 20 min.    -   (d) determining bacterial endotoxin in the sample and the PPC by        using a LAL assay.

In addition, as indicated above, it is envisaged in the herein providedendotoxin determination method that a “LER positive control” is preparedand used in step (d) to show the LER effect. Thus, a preferred aspect ofthe invention relates to the herein provided method for determiningbacterial endotoxin in a sample comprising an antibody exhibiting a LEReffect, wherein the method comprises the following steps in thefollowing order:

-   -   (a0) preparing a PPC by        -   spiking a known amount of endotoxin (e.g. to a final            concentration of 5.0 EU/ml) into a first aliquot of the            sample comprising an antibody, and        -   shaking the endotoxin spiked aliquot for 60 min to 2 hours            (preferably for 60 min at room temperature),    -   (a) adding magnesium ions, preferably in form of MgCl₂, to a        second aliquot of the sample as well as to the PPC to a final        concentration of 10-100 mM, preferably 40-75 mM, most preferably        45-55 mM; (and preferably incubating the sample and the PPC for        30 min to 6 hours, more preferably for 1-4 hours, most        preferably for 1 hour, (wherein the sample may be shaked before        and after the incubation)),    -   (b) diluting the sample and the PPC at a ratio of 1:5        (sample/PPC:buffer) to 1:20 (sample/PPC:buffer), preferably 1:10        (sample/PPC:buffer) with 10-50 mM Tris/HCl-buffer, pH 6.0-8.0,        preferably with 50 mM Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) and the PPC against endotoxin-free water        for 1-48 hours, preferably for 4-24 hours, most preferably for        24 hours. (Preferably, a cellulose acetate membrane with a        molecular weight cut-off of 10 kDa is used and the water is        changed after 2 and 4 hours. Most preferably, a Fast Spin        Dialyzer is used and the frequency of the stirrer is high.)        After dialysis, the samples and the PPC are shaked for 10 min to        1 hour, preferably for 20 min.    -   (d) Determining bacterial endotoxin in the sample and the PPC by        using a LAL assay, wherein in the LAL assay a “LER positive        control” is used, which is prepared by:        -   spiking CSE to a final concentration of 5.0 EU/ml to a third            aliquot of the sample to be tested;        -   shaking the spiked aliquot for ≥60 min, most preferably for            60 min;        -   diluting the spiked aliquot with endotoxin-free water (the            spiked aliquot is diluted to the same extend as the sample            to be tested in step (b) of the herein provided methods);        -   shaking the spiked aliquot (e.g. for 1 min).

Thus, a preferred aspect of the invention relates to the herein providedmethod for determining bacterial endotoxin in a sample comprising anantibody exhibiting a LER effect, wherein the method comprises thefollowing steps in the following order:

-   -   (a0) preparing a PPC by        -   spiking a known amount of endotoxin to a final concentration            of 5.0 EU/ml into a first aliquot of the sample comprising            an antibody, and        -   shaking the endotoxin spiked aliquot for about 60 min at            room temperature,    -   (a) adding magnesium ions, preferably in form of MgCl₂, to a        second aliquot of the sample and the PPC to a final        concentration of 45-55 mM, shaking the sample and the PPC for 1        min, incubating the sample and the PPC for 1 hour, and shaking        the sample and the PPC again after the incubation,    -   (b) diluting the sample and the PPC 1:10 (sample/PPC:buffer)        with 50 mM Tris/HCl-buffer, pH ˜7.0,    -   (c) dialyzing the sample having a pH-value of 5.7-8.0        (preferably 6.5-7.5) and the PPC against endotoxin-free water        for 24 hours by using a cellulose acetate membrane with a        molecular weight cut-off of 10 kDa; wherein the water is changed        after 2 and 4 hours (preferably, a Fast Spin Dialyzer is used        and the frequency of the stirrer is high), and shaking the        sample and the PPC for 20 min, and    -   (d) determining bacterial endotoxin in the sample and the PPC by        using a LAL assay, wherein in the LAL assay a “LER positive        control” is used, which is prepared by        -   spiking CSE to a final concentration of 5.0 EU/ml to a third            aliquot of the sample to be tested,        -   shaking the spiked aliquot for 60 min,        -   diluting the spiked aliquot at a ratio of 1:10 with            endotoxin-free water,        -   shaking the spiked aliquot (e.g. for 1 min).

In addition, as mentioned above, it is envisaged that water controls areapplied in the LAL assay. For example, a water control that consists ofendotoxin-free water may be applied in the LAL assay of step (d) of theherein provided endotoxin determination method. Another water controlmay consist of endotoxin spiked endotoxin-free water. After endotoxinspiking, the water is preferably shaked [e.g. in a Heidolph Multi Reaxshaker, high speed (2,037 rpm)] for ≥60 min (e.g. for 60 min at roomtemperature). In addition, in a LAL assay a standard is normallyprepared according to the instructions of the used kit.

Steps (a0), (a), (b), (c) and (d) are to be conducted in the order(a0)(a)(b)(c)(d). However, washing of the dialysis membrane can beperformed at any time, provided that the step is executed when thedialysis starts. Similarly, preparation of the LER positive control canbe performed at any time provided that the step is executed when the LALassay starts. In a preferred aspect of the invention, the hereinprovided endotoxin determination method comprises the following steps.

Step (a00): Preparation of the Samples

-   -   Adapting the concentration of the sample to be tested to the PPC        (e.g. antibody 900 μl+100 μl endotoxin-free water)    -   Spiking an aliquot of the sample to be tested with endotoxin for        the production of the PPC (e.g. antibody 900 μl+100 μl CSE conc.        50 EU/ml=final conc. 5.0 EU/ml)    -   Preparing a water control (e.g. endotoxin-free water 1000 μl)    -   Preparing another water control (e.g. endotoxin-free water 900        μl+100 μl CSE conc. 50 EU/ml=final conc. 5.0 EU/ml)    -   Shake the samples about 60 min at room temperature [e.g. in a        Heidolph Multi Reax shaker, high speed (2,037 rpm)],        Step (a01): Washing of the Dialysis Membrane    -   For example, use 10 kDa cellulose acetate (CA) membranes and put        them into a crystallizing dish with endotoxin-free water (e.g.        300 ml of distilled water of the manufacturer B. Braun,        Melsungen)    -   Shake them carefully for 1 h (Shaker SG 20. IDL GmbH, Germany or        equivalent, 50 to 300 rpm, preferably 100 rpm)    -   Transfer the membranes into an new crystallizing dish with fresh        endotoxin-free water (e.g. 300 ml of distilled water of the        manufacturer B. Braun, Melsungen)    -   Shake (Shaker SG 20. IDL GmbH, Germany or equivalent, 50 to 300        rpm, preferably 100 rpm) them for 1 h

Step (a): Addition of 25-100 mM, Preferably 50-100 mM, Magnesium Ions(Mg²)

-   -   Add Mg²⁺, e.g. in form of MgCl₂, to the samples of step (a0) to        a final concentration of 25-100 mM, preferably 50-100 mM (e.g.        add 50 μl of an 1 M MgCl₂ stock solution to the samples of step        (a0))    -   Shake [e.g. in a Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature] for 2-5 min, e.g. for 1 min    -   Incubate the samples for 45 to 75 minutes, preferably for 60        min, at room temperature    -   Shake [e.g. in a Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature] for 1 min

Step (b): Dilution

-   -   Take one of the samples of step (a) and dilute it 1:10 with        buffer pH ˜7.0 (e.g. 50 mM Tris/HCl buffer pH ˜7.0) (e.g. 895 μl        50 mM Tris-buffer+105 μl sample)    -   Preferably prepare two diluted samples    -   For example:        -   2×antibody 1:10 with Tris-buffer (sample)        -   2×antibody spiked with 5.0 EU/ml 1:10 with Tris-buffer (PPC)        -   2×LAL water 1:10 with Tris-buffer (background)        -   2×LAL water 5.0 EU/ml 1:10 with Tris-buffer (standard)        -   LAL-water=please add

Step (c): Dialysis

-   -   Shake [e.g. in a Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature, all diluted samples for 1 min    -   Transfer them into the dialyzer (preferably a FastSpinDIALYZER)    -   Put one dialyzer per beaker on a stirrer    -   Fill the beaker with endotoxin-free water (e.g. 200 ml of        distilled water of the manufacturer B. Braun, Melsungen)    -   Dialyze 24 h at room temperature and exchange the endotoxin-free        water after 2 h and 4 h    -   The frequency of the stirrer is preferably 50 to 300 rpm, more        preferably 200 rpm (especially if a FastSpinDIALYZER is used).        The stirrer has preferably a length of 20-60 mm and a diameter        of 5-25 mm. More preferably, the stirrer is a magnetic stirrer        having a length of about 40 mm and a diameter of about 14 mm.

Step (d): Shaking

-   -   After dialysis transfer the samples into new vessels (e.g. into        1.5 ml screw vials) and shake [e.g. in a Heidolph Multi Reax        shaker, high speed (2,037 rpm) at room temperature] for 20-60        min        Step (d00): Preparation of the “LER Positive Control” and of        Further Water Controls

-   1. (Step (d00) not necessarily has to be performed after step (d).    Step (d00) can be performed at any time provided that the “LER    positive control” and the further water controls are ready when the    LAL assay starts.) Prepare the “LER positive control” (i.e. the    “positive LER control”) preferably 1 h before the dialysis ends    [e.g. antibody 900 μl+100 μl CSE conc. 50 EU/ml=final conc. 5.0    EU/ml]    -   Prepare further water controls, for example:        -   1. antibody 900 μl+100 μl LAL water        -   2. LAL water 1000 μl    -   LAL water 900 μl+100 μl CSE conc. 50 EU/ml=final conc. 5.0 EU/ml    -   Shake 1 h [e.g. in a Heidolph Multi Reax shaker, high speed        (2,037 rpm) at room temperature]    -   Dilute samples 1:10 with endotoxin-free water    -   Shake for 1 min [e.g. in a Heidolph Multi Reax shaker, high        speed (2,037 rpm) at room temperature]

Step (e): LAL Assay

-   -   Prepare the standard (i.e. the standard that is comprised in the        used LAL assay kit) according to the instructions of the        manufacturer and start the measurement as follows;

(1) Preparation of LAL Reagent (Kinetic-QCL™ Reagent):

-   -   Reconstitute the co-lyophilized mixture of lysate prepared from        the amoebocytes of the horseshoe crab, Limulus polyphemus, and        chromogenic substrate with 2.6 ml of LAL Reagent Water per vial        immediately before use.        (2) Preparation of CSE Stock Solution (50 EU/Ml, i.e. Equivalent        to Standard S1):    -   Reconstitute the CSE preparation (E. coli O55:B5-LPS, each vial        contains 50-200 EU lyophilized endotoxin) in the volume of LAL        Reagent Water stated on the Certificate of Analysis and        calculated to yield a solution containing 50 EU(or IU)/ml.    -   Shake the CSE Stock solution vigorously for at least 15 minutes        at high speed on a shaker.    -   Prior to use, let the solution warm up to room temperature and        shake again vigorously at high speed on a shaker for 15 minutes.

(3) Preparation of CSE Standard Series:

-   -   Dilute the CSE stock solution/standard S1 (step 1) with LAL        Reagent Water at room temperature in a 1:10 scheme to yield the        complete series of CSE standards (50, 5, 0.5, 0.05 and 0.005        EU/ml

(4) LAL Analysis in a 96-Well Microplate ELISA Reader Format:

-   -   Carefully dispense 100 μl of the LAL Reagent Water blank,        endotoxin standards, product samples, positive product controls.        into the appropriate wells of the microplate.    -   Place filled plate in the microplate reader, close the lid.    -   Pre-incubate the plate for ≥10 minutes at 37° C.±1° C.    -   Using an 8-channel multipipettor dispense 100 μl of the        Kinetic-QCL™ Reagent into all wells of the microplate beginning        with the first column (A1-H1) and proceeding in sequence to the        last column used. Add reagent as quickly as possible (avoid air        bubbles).    -   Immediately click on the OK button on the computer keyboard to        initiate the test. (Note: The Kinetic-QCL™ assay is performed        with the microplate cover removed)

Herein, “spiking” means “adding” or “providing with”. For example,“spiking a sample with a known amount of CSE” means “adding a knownamount of CSE to a sample” or “providing a sample with a known amount ofCSE”.

Endotoxins, also known as lipopolysaccharides (LPS), are large moleculesfound in the outer membrane of Gram-negative bacteria, and elicit strongimmune responses in animals, e.g. in humans. As mentioned, the inventionprovides for a method for determining (i.e. detecting and quantifying)bacterial endotoxin in a sample comprising an antibody, wherein themethod comprises the herein described steps (a) to (d) (preferably alsoincluding the steps (a00), (a01) and (d00)).

In one embodiment the endotoxin may be Eschericha coli endotoxin.Accordingly, the endotoxin that is determined (i.e. detected and/orquantified) in step (d) of the herein provided endotoxin determinationmethod may be E. coli endotoxin. For example, the endotoxin that isspiked in the sample during the LAL assay may be E. coli endotoxin (i.e.endotoxin purified from E. coli). Preferably, the endotoxin is acommercially available E. coli endotoxin (e.g. control standardendotoxin, CSE).

The WHO International Standard Endotoxin (I.S.) is an endotoxinpreparation from E. coli O113:H10:K—that is internationally recognizedas the ultimate calibrant for the bacterial endotoxins test. The currentlot of the International Standard is termed “WHO International Standard,3rd I.S. for endotoxin”.

An Reference Standard Endotoxin (RSE) is an endotoxin preparation thathas been calibrated against the WHO International Standard Endotoxin.RSEs are established by national agencies (like USP, EP, JP, ChP) andprovided to calibrate CSEs (see below) for use in the LAL assays.

A Control Standard Endotoxin (CSE) is an endotoxin preparation otherthan RSE that has been calibrated against an RSE. CSEs arevendor-specific, highly-purified preparations of endotoxins that areproduced from E. coli O113:H10:K—(e.g. Associates of Cape Cod, Inc.) orother E. coli strains like E. coli O55:B5 (e.g. Charles River, Lonza).Vendors might add stabilizers like human serum albumin, PEG, or starchat their own discretion. CSEs are supplied in various concentrations,depending on their intended use.

The herein provided method for determining (i.e. detecting and/orquantifying) endotoxin in a sample comprising an antibody; or the hereinprovided method for the preparation of a sample comprising an antibodyhave the advantageous effect that they obviate the LER effect in the LALassay. Thus, one aspect of the invention relates to the use of theherein provided sample preparation method or the herein providedendotoxin determination method for overcoming the LER effect in thedetermination of bacterial endotoxin in a LAL assay.

More specifically, the herein provided sample preparation method or theherein provided endotoxin determination method have the advantageouseffect that these methods render the sample comprising an antibody thatexhibits a LER effect reactive to factor C in the LAL enzymatic cascade.Thus, one aspect of the invention relates to the use of the hereinprovided sample preparation method or the herein provided endotoxindetermination method for rendering the sample comprising an antibodyexhibiting a LER effect reactive to factor C in the LAL enzymaticcascade.

Herein the term “determining”, in particular in the context of“determining bacterial endotoxin” or grammatical variations thereofrelates to the detection and/or quantification of endotoxin, preferablyto the detection and quantification of endotoxin. In context of thepresent invention, endotoxin (e.g. E. coli endotoxin such as CSE) ispreferably determined by a LAL assay.

The term “bacterial endotoxins test” or “bacterial endotoxin test” areused interchangeously herein and relate to a group of tests to detect orquantify endotoxins from Gram-negative bacteria. The BET describes thecompendial (i.e. related to a compendium that serves as a standard, suchas the European or US Pharmacopeia, or other national or internationalpharmaceutical standard) LAL assay (limulous amoebocyte lysate assay).Moreover, in context of the invention it is preferred that the pH-valueof the sample to be tested during the LAL assay is from 5.7-8.0,preferably 6.0-8.0, more preferably 6.5-7.5.

The term “LAL assay” is commonly known in the art and represents an invitro endotoxin test for human and animal parenteral drugs, biologicalproducts, and medical devices. In particular, the LAL assay is a test todetect and quantify endotoxins from Gram-negative bacteria using theamoebocyte lysate from the horseshoe crab (Limulus polyphemus orTachypleus tridentatus). For example, during bacterial cellreproduction, cell division, vegetation dieback and cell lysis, LPSmolecules are released from the bacterial cell surface in a ratheruncontrolled and unspecific manner. The released LPS represent a potentbacterial toxin and is primarily responsible for the toxic manifestationof severe infections with Gram-negative bacteria and detrimental effects(e.g., high fever, hypotension and irreversible shock) (Rietschel, 1994,FASEB J. 8:217-225). The lipid A component is responsible for thisbiological activity of LPS. In diluted salt solutions, LPS formmacromolecular aggregates (micelles). The formation, size and dynamicsof these micelles is correlated to the LPS concentration, variousphysico-chemical parameters (such as temperature, concentration of thebuffer (ionic strength), and pH) as well as the structure of theO-chain, which is the core-oligosaccharide of lipid A (Aurell, 1998,Biochem. Biophys. Res. Comm. 253:119-123). The lipid A moiety of LPS,which is highly conserved among all Gram-negative bacteria, is that partof the LPS molecule that is recognized by the LAL assay, rendering thistest a golden standard and a suitable procedure to investigate endotoxincontamination from a broad entity of Gram-negative bacterial sources(Takada (1988) Eur. J. Biochem; 175:573-80).

The principles of the LAL assay are described as follows. In the LALassay the detection of LPS takes place via gelation of the LAL. This LALactivating activity of LPS is affected by a variety of factors:

-   -   the formation of LPS-LPS aggregates [Akama, 1984, In “Bacterial        Endotoxin” (Eds. J. Y. Homma, S. Kanegasaki, O. Lüderitz, T.        Shiba and O. Westphal], Publisher Chemie)    -   the formation of protein-LPS aggregates, e.g. with human        lipoproteins Apo A 1, lysozyme, ribonuclease A or human lgG        (Emancipator, 1992, Infect Immun. 60:596-601; Petsch, 1998,        Anal. Biochem. 259:42-47)    -   the method of extraction of LPS from bacterial cells [Galanos,        1984, In “Bacterial Endotoxin” (Eds. J. Y. Homma, S.        Kanegasaki, O. Lüderitz, T. Shiba and O. Westphal), Publisher        Chemie]    -   the bacteria species; the LAL activating activity within        Enterobacteriaceae varies by a factor of 1000 [Niwa, 1984, In        “Bacterial Endotoxin” (Eds. J. Y. Homma, S. Kanegasaki, O.        Lüderitz, T. Shiba and O. Westphal), Publisher Chemie].

The LAL assay is harmonized among the pharmacopeia in the United States(US), Europe (EP) and Japan (JP). In the harmonized pharmacopeiachapters (USP <85>, Ph. Eur. 2.6.14., and JP 4.01), three techniques forthe LAL assay are described:

-   -   gel-clot technique (based on an endotoxin-induced gelling)    -   turbidimetric technique (based on the turbidity induced by the        gelling)    -   chromogenic technique (based on the coloring after splitting of        a synthetic peptide-chromogen complex).

These three techniques are in turn applied in 6 different methods:

-   -   Method A: gel-clot method, limit test    -   Method B: gel-clot method, semi-quantitative test    -   Method C: kinetic turbidimetric method    -   Method D: kinetic chromogenic method    -   Method E: chromogenic end-point method    -   Method F: turbidimetric end-point method

Per Ph. Eur./USP/JP these six methods are to be viewed as equivalent.

A prioritized aspect of the present invention relates to the hereinprovided sample preparation method and the herein provided endotoxindetermination method, wherein the kinetic chromogenic method or thekinetic turbidimetric method is used for the determination of bacterialendotoxin in the sample. Most preferably, the kinetic chromogenic methodis used in the herein provided sample preparation method and endotoxindetermination method. By using this technique endotoxin can be detectedphotometrically. This technique is an assay to measure the chromophorereleased from a chromogenic substrate (i.e. a suitable chromogenicpeptide) by the reaction of endotoxins with LAL. The kinetic chromogenicassay is a method to measure either the time (onset time) needed toreach a predetermined absorbance of the reaction mixture, or the rate ofcolor development. The test is carried out at the incubation temperaturerecommended by the lysate manufacturer (which is usually 37±1° C.). Forexample, for performing the kinetic chromogenic LAL assay, a sample maybe mixed with a reagent comprising LAL and a chromogenic substrate (i.e.a suitable chromogenic peptide such as Ac-Ile-Glu-Ala-Arg-pNA and placedin an incubating plate reader. Then, the sample is monitored over timefor the appearance of a color (e.g. a yellow color). The time requiredbefore the appearance of a color (reaction time) is inverselyproportional to the amount of endotoxin present. That is, in thepresence of a large amount of endotoxin the reaction occurs rapidly; inthe presence of a smaller amount of endotoxin the reaction time isincreased. The concentration of endotoxin in unknown samples can becalculated from a standard curve. During the LAL assay, i.e. in step (d)of the herein provided endotoxin determination method, thequantification of endotoxin is preferably carried out via a standardcalibration curve, which covers a range of at least two orders ofmagnitude (in one aspect of the invention 0.005, 0.05, 0.5, 5.0 and 50.0EU/ml).

For example, during the kinetic chromogenic LAL technique, the followingreactions may take place. Gram negative bacterial endotoxin catalyzesthe activation of a proenzyme in the LAL. The initial rate of activationis determined by the concentration of endotoxin present. The activatedenzyme catalyzes the splitting of p-nitroaniline (pNA) from thecolorless substrate Ac-Ile-Glu-Ala-Arg-pNA. The pNA released is measuredphotometrically, at 405 nm continuously throughout the incubationperiod. The concentration of endotoxin in a sample is calculated fromits reaction time by comparison to the reaction time of solutionscontaining known amounts of endotoxin standard. For the LAL assay, thekit “Limulus Amoebocyte Lysate (LAL) Kinetic-QCL™” from LONZA (CatalogNumber: 50-650U, 50-650NV, 50-650H; K50-643L, K50-643U) may be usedaccording to the instructions of the manufacturer. By performing the LALassay it is envisaged to use the endotoxin, which is comprised in theused kit (e.g. E. coli O55:B5 Endotoxin, which is comprised in the kit“Limulus Amoebocyte Lysate (LAL) Kinetic-QCL™” from LONZA, CatalogNumber: 50-650U, 50-650NV, 50-650H; K50-643L, K50-643U).

In context of the invention it is preferred that the pH-value of thesample to be tested during the LAL assay is from 5.7-9.0. Morepreferably, the pH-value of the sample to be tested during the LAL assayis from 5.8-8.0, even more preferably from pH 5.8-7.5, even morepreferably from pH 5.8-7.0. Most preferably, the pH-value of the sampleto be tested during the LAL assay is from 5.8-7.0. For example, thepH-value of the sample to be tested during the LAL assay may be pH 5.7,pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6,pH 6.7, pH 6.8, pH 6.9, or pH 7.0. Thus, it is envisaged in context ofthe invention that before the LAL assay the pH value of the testsolution (i.e. a dissolved solid sample or liquid sample) is adjusted tobe between pH 5.7-8.0, more preferably between pH 5.8 and pH 7.0. Ifnecessary, the pH value is to be adjusted e.g. by dilution, addition ofbuffers and/or neutralization.

Several substances (such as β-glucans) interfere with the LAL test tosome degree (the obvious exception being water samples). Interferencecan be inhibition or enhancement of the LAL assay. In particular,interference factors may either enhance or diminish the LPSquantification obtained from the LAL test, and therefore thequantification of the endotoxin. Therefore, if in step (d) of the hereinprovided endotoxin determination method the recovery of the PPC is notin the acceptable range of 50-200%, the interference factor must beremoved. This can be done by sample dilution in step (b) of the hereinprovided method. In particular, the sample may be diluted withendotoxin-free water or endotoxin-free buffer (preferably with Tris/HClbuffer, pH ˜7.0). The lowest sample dilution (highest productconcentration) that lacks inhibition/enhancement is called“non-interfering concentration (NIC)”. However, during sample dilution,the MVD (Maximum Valid Dilution=maximum possible dilution of a sample inwhich an endotoxin limit can be determined) may not be exceeded. Inparticular, based on the test results of the different batches, a sampledilution is chosen that covers all batches (validated sample dilution orsample concentration). Or, in other words, the sample dilution thatresults in a recovery of 50-200% in the PPC is chosen in step (b) of theherein provided methods. To establish that the treatment choseneffectively eliminates interference without loss of endotoxins (i.e.without showing the LER effect) the “Test for Interfering Factors” canbe performed by using a sample that is spiked with a definedconcentration of endotoxin (i.e. a PPC).

Accordingly, one aspect of the invention relates to the herein providedendotoxin determination method, wherein a PPC is prepared and tested forendotoxin in step (d) of the herein provided endotoxin determinationmethod. The sample is free of interfering factors if the recovery of thespiked endotoxin control standard amounts to 50-200%.

Due to the fact that the BET per USP/Ph. Eur./JP includes an internalcontrol (PPC) that allows assessment of each test result individually,BET method validation per USP/Ph. Eur./JP is not a prerequisite forcorrect endotoxins results.

The term “low endotoxin recovery (LER)” or “LER effect” is known in theart and describes endotoxin masking specifically caused by a combinationof polysorbate plus either citrate or phosphate (Chen, J. and Williams,K. L., PDA Letter 10, 2013, 14-16). Endotoxin masking may also be causedby any other buffer component or combinations thereof. To identify thata given material (e.g. a buffer or a sample of a therapeutic antibody)exhibits the LER effect, endotoxin contents can be monitored over time,e.g. in an endotoxin hold time study. Endotoxin hold time studiesrequire endotoxin spiking of an undiluted sample and storage of theendotoxin spiked sample over time. For example, the sample may be storedup to several. Preferably, in a hold time study the endotoxin spikedsample is stored for several (e.g. 7 for up to 28) days and at definedtime points a LAL assay is performed. Recovery rates that are lower than50% of the amount of the spiked endotoxin indicate that the sampleexhibits a LER effect. If the endotoxin recovery is less than 50% butonly occurs in any of the middle time points but not the end timepoints, the test sample cannot be considered to exhibit a maskingeffect.

During the last years, FDA has well recognized the LER phenomenon andissued guidance (see Hughes, P., et al., BioPharm. Asia March/April2015, 14-25). These guidance define the acceptable limits of endotoxinrecovery in pharmaceutical specimens to range between 50 and 200% once adefined amount of CSE was spiked to the undiluted sample before (e.g.5.0 EU/ml=100%). In case a sample to be tested exhibits the LER effect,the recovery rate of the spiked endotoxin is below 50% of the totalamount of the spiked endotoxin.

In the inventive methods provided herein, the sample comprises anantibody, preferably a monoclonal antibody. Herein the terms “sample”,“sample to be tested”, “sample comprising an antibody” and “samplecomprising an antibody to be tested” are used interchangeously and referto a certain amount of liquid comprising an antibody that is to betested for the presence and/or amount of endotoxin. Or, in other words,the terms “sample”, “sample to be tested”, “sample comprising anantibody” and “sample comprising an antibody to be tested” are usedinterchangeously herein and relate to a liquid to be tested for thepresence and/or amount (preferably for the presence and amount) ofendotoxin, wherein said liquid comprises an antibody. Said “samplecomprising an antibody to be tested” is preferably a sample of atherapeutic antibody. The term “therapeutic antibody” relates to anyantibody preparation that is intended for use in a human being. Theantibody (e.g. the therapeutic antibody) is preferably formulated withpolysorbate 80 or sodium citrate buffer, more preferably withpolysorbate 80 and sodium citrate buffer. Most preferably, the antibodyis formulated with an about 25 mM sodium citrate buffer and about 700mg/L polysorbate 80 and has a pH value of about 6.5. It is preferred incontext of the present invention that said antibody (e.g. thetherapeutic antibody) is a monoclonal antibody. Most preferably, saidantibody (e.g. the therapeutic antibody) is the anti-CD20 antibodyrituximab. Thus, in context of the invention, the sample may be a sampleof MabThera®/Rituxan®/Zytux®. It is envisaged in context of theinvention that the sample to be tested (i.e. the sample comprising anantibody to be tested) shows/exhibits the LER effect.

Herein the term “antibody” is used in the broadest sense andspecifically encompasses intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies)formed from at least two intact antibodies, and antibody fragments, solong as they exhibit the desired biological activity. Also human,humanized, camelized or CDR-grafted antibodies are comprised.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies of the population of antibodies are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe constructed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies that are comprised in thesample of the methods of the present invention may be made by thehybridoma method first described by Kohler, G. et al., Nature 256 (1975)495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).

The monoclonal antibodies described herein are preferably produced byexpression in a host cell, most preferably a Chinese hamster ovary (CHO)cell. For production isolated nucleic acid encoding an the antibodyencoding an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychains of the antibody) is inserted in one or more vectors (e.g.,expression vectors). These are introduced into host cell. The host cellcomprises (e.g., has been transformed with): (1) a vector comprising anucleic acid that encodes an amino acid sequence comprising the VL ofthe antibody and an amino acid sequence comprising the VH of theantibody, or (2) a first vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and a secondvector comprising a nucleic acid that encodes an amino acid sequencecomprising the VH of the antibody. The host cell can be eukaryotic, e.g.a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20cell).

For recombinant production of antibody, nucleic acid encoding theantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.No. 5,648,237, U.S. Pat. No. 5,789,199, and U.S. Pat. No. 5,840,523.(See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248,Lo, B. K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254,describing expression of antibody fragments in E. coli.) Afterexpression, the antibody may be isolated from the bacterial cell pastein a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified that may be used in conjunctionwith insect cells, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat. No. 6,420,548, U.S.Pat. No. 7,125,978, and U.S. Pat. No. 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR−CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

“Antibody fragments” comprise a portion of an intact antibody. The term“antibody fragments” includes antigen-binding portions, i.e., “antigenbinding sites” (e.g., fragments, subsequences, complementaritydetermining regions (CDRs)) that retain capacity to bind an antigen(such as CD20), comprising or alternatively consisting of, for example,(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region;(iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward; 1989; Nature 341; 544-546), whichconsists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Antibody fragments or derivatives furthercomprise F(ab′)2, Fv or scFv fragments or single chain antibodies.

Preferably, in the herein provided methods the antibody (i.e. theantibody that is comprised in the sample) is rituximab.

The term “rituximab” (trade names MabThera®, Rituxan®, Zytux®) relatesto a chimeric monoclonal antibody against the protein CD20. CD20 isfound on the surface of cancerous and normal B-cells. Rituximab destroysB cells and is therefore used, e.g., to treat diseases that arecharacterized by excessive numbers of B cells, overactive B cells, ordysfunctional B cells. This includes many lymphomas, leukemias,transplant rejection, and autoimmune disorders. For example, rituximabis used in chronic lymphocytic leukemia as a subcutaneous formulation.However, rituximab is usually administered by intravenous infusion. Stemcells in bone marrow do not have the CD20 protein allowing B-cells torepopulate after rituximab treatment. As used herein, the term“rituximab” also encompasses all anti-CD20 antibodies or anti-CD20antibody fragments that fulfil the requirements necessary for obtaininga marketing authorization in a country or territory selected from thegroup of countries consisting of the USA, Europe and Japan. Mostpreferably, the term “rituximab” refers to an antibody having the aminoacid sequences of the heavy and light chain as shown in SEQ ID NOs: 1and 2, respectively. The person skilled in the art readily knows how toobtain a coding nucleic acid sequence from a given amino acid sequence.Thus, with the knowledge of SEQ ID NOs: 1 and 2, coding nucleic acidsequences of rituximab can easily be obtained.

The trade name “NeoRecormon” refers to a pharmaceutical formulation thatcontains as active ingredient epoetin beta. Epoetin beta is a syntheticversion of the naturally-occurring hormone erythropoietin.Erythropoietin is produced by healthy kidneys and stimulates the bonemarrow to produce red blood cells, which carry oxygen around the body.Epoetin beta is also used to treat symptomatic anaemia in people withcertain types of cancer who are having chemotherapy. One of the sideeffects of chemotherapy is that it kills healthy blood cells as well ascancer cells. Injections of epoetin increases red blood cell productionand helps relieve the symptoms of anaemia. As epoetin increases bloodcell production, a larger volume of blood can be taken from peoplereceiving epoetin and this blood can be stored for transfusion during orafter the surgery.

In step (d) of the herein provided sample preparation or endotoxindetermination method, the sample (i.e. the sample comprising anantibody) is dialyzed against an endotoxin-free aqueous solution,wherein the sample has a pH-value between pH 5.7 and pH 8.0 (preferablybetween pH 6.0 and 8.0, more preferably between 6.5 and 7.5). Inbiochemistry, dialysis is a commonly used process of separatingmolecules in solution by the difference in their rates of diffusionthrough a semipermeable membrane, such as dialysis tubing. Dialysis is acommon laboratory technique that operates on the same principle asmedical dialysis. In the context of life science research, the mostcommon application of dialysis is the removal of unwanted smallmolecules such as salts, reducing agents, or dyes from largermacromolecules such as antibodies. Dialysis is also commonly used forbuffer exchange and drug binding studies.

Diffusion is the random, thermal movement of molecules in solution(Brownian motion) that leads to the net movement of molecules from anarea of higher concentration to an area of lower concentration untilequilibrium is reached. In dialysis, a sample and a buffer solution(called the dialysate) are separated by a semi-permeable membrane thatcauses differential diffusion patterns, thereby permitting theseparation of molecules in both the sample and dialysate. Due to thepore size of the membrane, large molecules in the sample (e.g.antibodies) cannot pass through the membrane, thereby restricting theirdiffusion from the sample chamber. By contrast, small molecules (e.g.the components of a Na-citrate buffer) will freely diffuse across themembrane and obtain equilibrium across the entire solution volume,thereby changing the overall concentration of these molecules in thesample and dialysate. Once equilibrium is reached, the finalconcentration of molecules is dependent on the volumes of the solutionsinvolved, and if the equilibrated dialysate is replaced (or exchanged)with fresh dialysate (see procedure below), diffusion will furtherreduce the concentration of the small molecules in the sample.

For example, the following dialysis procedure for removing Na-citratebuffer from the sample (i.e. from the sample comprising an antibody) maybe used:

1. Obtaining and washing a membrane with a molecular weight cut-off of10 kDa2. Loading the sample into dialysis tubing, cassette or device3. Placing the sample into an external chamber with dialysate (withstirring of the buffer)4. Dialyzing for 24 hours at room temperature; changing water twiceduring said 24 hours

By using the appropriate volume of dialysate and multiple exchanges ofthe buffer, the concentration of the sodium citrate buffer within thesample can be decreased to negligible levels (i.e. 1-2% of the originalcontent).

The present invention is further described by reference to the followingnon-limiting figures and examples. In the Figures as well as in theExamples, most of the described experiments are indicated by definednumbers. For example, the designation [rituximab 117] means that theexperiment was performed with formulated rituximab and/or withformulated rituximab placebo and has the reference number “117”.

DESCRIPTION OF THE FIGURES

FIG. 1 Time dependency of dialysis of NeoRecormon containing phosphateand polysorbate 20 by using a MWCO of 12-16 kDa. Shown is the weight ofthe inner dialyzate obtained after the indicated dialysis time at roomtemperature, lyophilization and weighting. Data on top of gray bars showthe average amount of 2 measurements (%).

FIG. 2 Dialysis of NeoRecormon® containing phosphate and polysorbate 20by using a membrane MWCO of 12-16 kDa treated with or without (w/o)bovine serum albumin (BSA) prior to dialysis. Shown is the content ofphosphate (P) in the inner dialyzate obtained after the time indicated.Left bars show the amount of P when the membrane was treated with 0.2%BSA before dialysis. Right bars correspond to the amount of P withoutBSA-treatment. The photometric test of the phosphate recovered from theinner dialyzate was performed according to Strominger (1959, J. Biol.Chem. 234: 3263-3267).

FIG. 3: [Rituximab 115] and [Rituximab 117] Recovery rates (%) obtainedby performing the protocol for overcoming the LER effect as described inExample 2.1 by using (A) Rituximab and (B) Rituximab placebo as sample.In the Figures “fast spin” and “slow spin” means the frequency of thestirrer (i.e. “fast spin” means that the frequency of the stirrer ishigh). This exemplary protocol is also useful for routine qualitycontrol of other samples, preferably for specimen containing sodiumcitrate buffer and polysorbate 80 as detergent.

FIG. 4: Schematic representation of a modified protocol for overcomingthe LER effect and recovery rates obtained by performing said protocol.(A) Schematic representation of a protocol according to the inventionfor overcoming the LER effect (e.g. in rituximab and rituximab placebo).The detailed protocol is described in Example 2.2. This exemplaryprotocol is also useful for routine quality control of other samples,preferably for specimen containing sodium citrate buffer and polysorbate80 as detergent. (B) [rituximab 046] Recovery rate (%) of rituximab andrituximab placebo obtained by the LER assay after performing theprotocol according to FIG. 4(A). For further details, see protocoldescribed in Example 2.2.

FIG. 5: [Rituximab 059] Recovery rates (%) obtained by performing theprotocol as described in Reference Example 2 by using Rituximab assample.

FIG. 6: Recovery rates (%) obtained by performing the protocol asdescribed in Reference Example 3 by using Rituximab as sample. Recoveryrates obtained by performing the protocol as described in ReferenceExample 2.2 [rituximab 061].

FIG. 7: Recovery rates (%) obtained by performing the protocol asdescribed in Reference Example 4 by using Rituximab as sample. (A)Recovery rates obtained by performing the protocol as described inReference Example 3.1 [rituximab 062]. (B) Recovery rates obtained byperforming the protocol as described in Reference Example 3.2 [rituximab063].

FIG. 8: Recovery rates (%) obtained by performing the protocol asdescribed in Reference Example 5 by using Rituximab as sample. (A)Recovery rates obtained by performing the protocol as described inReference Example 4.1 [rituximab 064]. (B) Recovery rates obtained byperforming the protocol as described in Reference Example 4.2 [rituximab065].

FIG. 9: Recovery rates (%) obtained by performing the protocol asdescribed in Reference Example 6 by using rituximab and rituximabplacebo as sample. [rituximab 072].

FIG. 10: Recovery rates (%) obtained by performing the protocol asdescribed in Reference Example 7 by using rituximab and rituximabplacebo as sample. (A) [rituximab 079] no incubation; (B) [rituximab080] 4 h incubation; (C) [rituximab 081] 1 day incubation; (D)[rituximab 082] 3 days incubation.

FIG. 11: Recovery rates (%) obtained by performing the LAL assay asdescribed in Reference Example 8 by using Rituximab as sample. (A)[rituximab 002] LAL assay with different dilutions; (B) [rituximab 004]comparison of Lonza and ACC CSE spiking; (C) [rituximab 005] LAL assaywith different dilutions and pH adjustment.

FIG. 12: Recovery rates (%) obtained by performing the LAL assay asdescribed in Reference Example 8 by using Rituximab as sample. Dialysisand dilution alone does not overcome the LER effect [rituximab 011].

FIG. 13: Time dependency of the LER effect. The Figure shows recoveryrates (%) obtained by performing spiking and the LAL assay as describedin Reference Example 1 by using Rituximab as sample [rituximab 027]. Theshaking time after spiking (i.e. 2 sec to 60 min) is indicated.

FIG. 14: Importance of the buffer system on the LER effect. Recoveryrates (%) obtained by performing the LAL assay as described in ReferenceExample 10 are shown. (A) LAL assay when Rituximab or sodium citrate areused as sample and diluted at a ratio of 1:2, 1:5, 1:10, or 1:20[rituximab 006]; (B) LAL assay when sodium citrate; polysorbate 80 orsodium citrate and polysorbate 80 are used as sample and diluted at aratio of 1:2, 1:5 or 1:10 [rituximab 029].

FIG. 15: Effect of MgCl₂ on the LER effect. Recovery rates (%) obtainedby performing the LAL assay as described in Reference Example 13 areshown. (A) Addition of MgCl₂ to a concentration of 10 mM [rituximab030]; (B)Addition of MgCl₂ to a concentration of 50 mM [rituximab 031];(C) Addition of MgCl₂ to a concentration of 25 mM [rituximab 032]; (D)Addition of MgCl₂ to a concentration of 75 mM [rituximab 033].

FIG. 16: Effect of mechanical treatments on the LER effect. Recoveryrates (%) obtained by performing the LAL assay as described in ReferenceExample 14 are shown [rituximab 034]. In the Figure, “shaken” meansshaken for 60 min.

The following Examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example 1: Technical Equipment and Reagents 1. Technical Equipment 1.1Microplate Reader System (Herein Also Designated as “Reader”)

-   -   Infinite® 200 PRO, Multimode Microplate Reader; Tecan,        Switzerland/Tecan Deutschland GmbH, Germany, P/N: 30050303.    -   Magellan V. 7.1 Software    -   Costar™ Cell Culture Plates, 96 Wells, Fisher Scientific, P/N:        07-200-89.

1.2 Shaker System and Glass Vials

-   -   Multi Reax; Heidolph, Germany, P/N: 545-10000-00.    -   1.5 ml Screw Neck Glass Vials (N8); Macherey-Nagel GmbH & Co.        KG, Germany, P/N: 702004 (Qty. of 100).    -   N 8 PP screw cap, black, closed top; Macherey-Nagel GmbH & Co.        KG, Germany, P/N: 70250 (Qty. of 100).    -   4 ml Screw Neck Glass Vials (N13); Macherey-Nagel GmbH & Co. KG,        Germany, P/N: 702962 (Qty. of 100).    -   N 13 PP screw cap, black, closed top; Macherey-Nagel GmbH & Co.        KG, Germany, P/N: 702051 (Qty. of 100).

1.3 Dialysis Equipment

-   -   SpinDIALYZER™, chamber volume 1000 μl; Harvard Apparatus,        U.S.A., P/N 740314 (Qty. of 1) and 740306 (Qty. of 5), local        distributor: Hugo Sachs Elektronik Harvard Apparatus, GmbH,        Germany, P/N SP1 74-0306 (Qty. of 5). Remark: Use of Lot No:        032613.    -   The dialyzer is a simple single-sided device for dialysis of        biological samples. A broad range of dialyzer sizes are        available to accommodate sample volumes ranging from 20 μl to        5 ml. The catalogue Nb. for 1 ml (as used herein) is 74-0314.        The MWCO of the membrane ranges from 100 to 300,000 Da. The        entire unit is constructed of PTFE, a virtually unreactive        material.    -   Fast SpinDIALYZER, chamber volume 1000 μl, Harvard Apparatus,        U.S.A., P/N 740510 (Qty. of 1) or 740504 (Qty. of 5), Remark:        Two-sided membrane system, top plus bottom membrane.    -   The dialyzer is a reusable sample chamber made of PTFE for high        sample recovery and has been redesigned to provide larger        membrane surface areas for an even faster dialysis rate. The        Ultra-Fast Dialyzers are of 50 μl to 1500 μl volume and have        been used here with 1000 μl. The catalogue Nb. for 1 ml (as used        herein) is 74-0412.    -   Cellulose acetate membranes, 500 Da MWCO, Harvard Apparatus,        U.S.A., P/N: SP1 7425-CA500, local distributor: Hugo Sachs        Elektronik Harvard Apparatus GmbH, Germany, P/N: SP1 7425-CA500.    -   Cellulose acetate membranes, 10 kDa MWCO, Harvard Apparatus,        U.S.A., P/N: SP1 7425-CA10K, local distributor: Hugo Sachs        Elektronik Harvard Apparatus GmbH, Germany, P/N: SP1 7425-CA10K.        -   Remark: Tested in addition to the ‘standard’ 500 Da MWCO            membranes in LER investigations on Rituximab as well as in            the LER experiments on NeoRecormon®.    -   Cellulose acetate membranes, 25 kDa MWCO, Harvard Apparatus,        U.S.A., P/N:SP1 7425-CA25K, local distributor: Hugo Sachs        Elektronik Harvard Apparatus GmbH, Germany, P/N: SP1 7425-CA25K.        -   Remark: Tested in addition to the ‘standard’ 500 Da MWCO            membranes in LER investigations on Rituximab as well as in            the LER experiments on NeoRecormon®.    -   Aqua B. Braun, sterile pyrogen-free water, 1 l, B. Braun        Melsungen AG, Germany, P/N: 14090586.    -   Crystallizing Dishes, 900 ml, OMNILAB, Germany, P/N: 5144008.        (Remark: Use for rinsing of dialysis membranes)    -   DURAN® Beakers, tall form, 2000 ml, OMNILAB, Germany, P/N:        5013163.    -   DURAN® Beakers, tall form, 250 ml, OMNILAB, Germany, P/N:        5013136.

1.4 Routine Laboratory Equipments

-   -   Autoclaving System (Remark: Use for sterilisation of dialyses        chambers)    -   epT.I.P.S.® LoRetention-Reloads, PCR clean, 0.5-10 μl,        Eppendorf, Germany, P/N: 0030072.057    -   epT.I.P.S.® LoRetention-Reloads, PCR clean, 2-200 μl, Eppendorf,        Germany, P/N: 0030072.022    -   epT.I.P.S.® LoRetention-Reloads, PCR clean, 50-1000 μl,        Eppendorf, Germany, P/N: 0030072.030    -   Stripettes®, Individual, 5 ml, Paper/Plastic Wrap, Fisher        Scientific, P/N: 10420201.

2. Reagents 2.1 Kinetic Chromogenic LAL Assays and LAL-AssociatedReagents

-   -   Kinetic-QCL™ Kit; Lonza, Switzerland, P/N: 50-650U or 50-650H        (i.e. “Lonza kit”).    -   CHROMO—LAL von Associates of Cape Cod (AAC) Inc., USA, P/N:        C0031-5 (i.e. “ACC kit”).    -   Endotoxin E. coli O55:B5 for K-QCL; Lonza, Switzerland, P/N:        E50-643.    -   Endotoxin E. coli O55:B5, 2.5 mg/vial; Lonza, Switzerland, P/N:        N185.    -   LAL Reagent Water—100 ml; Lonza, Switzerland, P/N: W50-100.    -   MgCl₂, 10 mM solution for use with LAL, 30 ml vial; Lonza,        Switzerland, P/N: S50-641.    -   Magnesium chloride hexahydrate for analysis EMSURE® ACS, ISO,        Reag. Ph. Eur., 250 g; Merck, Germany, P/N: 1.05833.0250.    -   Tris buffer, 50 mM solution for use with LAL, 30 ml vial; Lonza,        Switzerland, P/N: S50-642.

2.2 Protein Reagents

-   -   Albumin bovine Fraction V, very low endotoxin, fatty acid free,        25 g; Serva, Germany, P/N: 47299.04.    -   Albumin, human serum, fraction V, high purity; 1 g; Merck,        Germany, P/N: 126658-1GM.

3. Tested Pharmaceuticals

For the herein described Examples, Rituximab (which comprises) andNeoRecormon® (which comprises epoetin-beta) were used. In addition, therespective placebos of Rituximab and NeoRecormon® were also applied inthe herein described methods.

The placebo of the respective sample is identical to the sample exceptfor the absence of the active therapeutic ingredient, i.e. rituximabplacebo does not contain rituximab but all other component of theformulation.

Example 2: Methods of the Invention for Overcoming the LER EffectExample 2.1: Protocol for Overcoming the LER Effect

In this Example rituximab and rituximab placebo were used as sample.However, as discussed below, the herein described protocol is useful forovercoming the LER in all typical formulations of pharmaceuticalantibodies.

Materials Used for this Example

Membranes:

-   -   10 kDa cellulose acetate (CA) membranes from Harvard Apparatus,        U.S.A., P/N:SP1 7425-CA10K

Dialyzer:

-   -   FastSpinDIALYZER, chamber volume 1000 μl, Harvard Apparatus,        U.S.A., P/N 740510 (Qty. of 1) or 740504 (Qty. of 5)

Sample Vials:

-   -   1.5 ml Screw Neck Glass Vials (N8); Macherey-Nagel GmbH & Co.        KG, Germany, P/N: 702004    -   N 8 PP screw cap, black, closed top; Macherey-Nagel GmbH & Co.        KG, Germany, P/N: 70250

Crystallizing Dishes:

-   -   900 ml, Duran, VWR Germany, P/N: 216-1817

MgCl₂ Stock Solution:

-   -   1M MgCl₂ dissolved in water (Magnesium chloride hexahydrate for        analysis EMSURE® ACS, ISO, Reag. Ph. Eur., 250 g; Merck,        Germany, P/N: 1.05833.0250)    -   Tris-Buffer, 50 mM solution for use with LAL (i.e.        endotoxin-free), 30 ml vial; Lonza, Switzerland, P/N: S50-642

Samples:

-   -   rituximab placebo and LAL water

Step by Step Protocol: Step 1: Preparation of the Samples

-   -   1×rituximab placebo 900 μl+100 μl LAL water    -   1×rituximab placebo 900 μl+100 μl CSE conc. 50 EU/ml=final conc.        5.0 EU/ml    -   1×LAL water 1000 μl    -   1×LAL water 900 μl+100 μl CSE conc. 50 EU/ml=final conc. 5.0        EU/ml    -   Shake the samples 60 min at RT (room temperature) [i.e. in a        Heidolph Multi Reax shaker, high speed (2,037 rpm)],

Step 2: Washing of Dialysis Membrane

-   -   Use ten 10 kDa cellulose acetate (CA) membranes and put them        into the crystallizing dish with 300 ml Aqua Braun (i.e.        distilled water of the manufacturer B. Braun, Melsungen)    -   Shake them for 1 h (Shaker SG 20. IDL GmbH, Germany or        equivalent, 50 to 300 rpm, preferably 100 rpm)    -   Transfer the membranes into an new crystallizing dish with fresh        Aqua Braun (also 300 ml)    -   Shake them for 1 h (Shaker SG 20. IDL GmbH, Germany or        equivalent, 50 to 300 rpm, preferably 100 rpm)        Step 3: Addition of MgCl₂ to a Final MgCl₂ Concentration of        about 50 mM MgCl₂    -   Add 50 μl of the 1M MgCl₂ stock solution to the samples of step        1    -   Shake them 1 min. [i.e. in a Heidolph Multi Reax shaker, high        speed (2,037 rpm) at room temperature]    -   Incubate the samples for 60 min at room temperature    -   Shake them 1 min. [i.e. in a Heidolph Multi Reax shaker, high        speed (2,037 rpm) at room temperature]

Step 4: Dilution

-   -   Take one of the samples of step 3 and dilute it 1:10 with        buffer, pH ˜7 (i.e. 50 mM Tris/HCl buffer pH ˜7)    -   895 μl 50 mM Tris-buffer+105 μl sample    -   Perform it twice for a repeat determination (i.e. determination        in duplicates):    -   2×rituximab placebo 1:10 with Tris-buffer    -   2×rituximab placebo 5.0 EU/ml 1:10 with Tris-buffer    -   2×LAL water 1:10 with Tris-buffer    -   2×LAL water 5.0 EU/ml 1:10 with Tris-buffer

Step 5: Dialysis

-   -   Shake all diluted samples for 1 min [i.e. in a Heidolph Multi        Reax shaker, high speed (2,037 rpm) at room temperature],    -   Transfer into the FastSpinDIALYZER    -   Put one dialyzer per beaker (i.e. DURAN® baker, tall form, 2000        ml, OMNILAB, Germany, P/N: 5013163) on a magnetic stirrer plate.        Adjust the frequency of the stirrer to be high (i.e. “fast        spin”). A high frequency of the stirrer means 50-300 rpm,        preferably 200-300 rpm. The stirrer is a heat-sterilized (4        hours at 250° C.) magnetic stirrer having a length of about 40        mm and a diameter of about 14 mm.

Fill the beaker with 200 ml Aqua Braun

-   -   Dialyze 24 h and exchange the Aqua Braun after 2 h and 4 h at        room temperature (21±2° C.)    -   After dialysis transfer the sample into new 1.5 ml screw vials

Step 6: Shaking

-   -   Shake the samples for 20 min. [i.e. in a Heidolph Multi Reax        shaker, high speed (2,037 rpm) at room temperature]

Step 7: Preparation of the LER Positive Control (i.e. the Positive LERControl) and of Further Water Controls

-   -   Prepare the LER positive control 1 h before the dialysis ends        -   1. rituximab placebo 900 μl+100 μl LAL water        -   2. rituximab placebo 900 μl+100 μl CSE conc. 50 EU/ml=final            conc. 5.0 EU/ml        -   3. LAL water 1000 μl        -   4. LAL water 900 μl+100 μl CSE conc. 50 EU/ml=final conc.            5.0 EU/ml    -   Shake 1 h [i.e. in a Heidolph Multi Reax shaker, high speed        (2,037 rpm) at room temperature]    -   Dilute samples 1:10 (sample:LAL water) with LAL water    -   Shake [i.e. in a Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature] for 1 min

Step 8: LAL Assay

-   -   Prepare the standard and start the LAL assay according to the        instructions of the manufacturer (Kinetic-QCL™ assay; Lonza)

Results and Discussion

As can be seen in FIGS. 3(A) (i.e. [rituximab 117]) and 3(B) (i.e.[rituximab 115]) the above described method is able to overcome the LEReffect. In addition, by using this method, the LER effect can beovercome in rituximab as well as in rituximab placebo. This indicatesthat the above described protocol in not dependent on a formulationcomprising a particular monoclonal antibody but can be used to obviatethe LER effect in every formulation comprising polysorbate 80 and achelating buffer (such as sodium citrate). This formulation is typicalfor antibodies, in particular monoclonal antibodies. Thus, the abovedescribed method is expected to be useful to overcome the LER effect inevery antibody formulation.

It has been found that Mg²⁺ is the divalent cation of choice to restoreLAL reactivity in formulations containing chelating buffers (such assodium citrate) and showing the LER effect.

In order to remove the chelating buffer (e.g. the Sodium citratebuffer), a second step (after addition of Mg²⁺) is to perform dialysis.The spinDIALYZER™ of Harvard is the preferred equipment for thedialysis.

The detergent (e.g. polysorbate 80) represents the second reason for theLER effect. In general, the presence of detergents (such as polysorbate80) in a biological sample leads to micelle formation in case thecritical micellar concentration (CMC) of the detergent (usually in theμM range) is reached. Micelles may inhibit the LPS-mediated activationof factor C, a serine protease representing the first enzyme in theLAL-cascade reaction (Nakamura (1988a) J. Biochem. 103: 370-374). Inmonoclonal antibody preparations, the undiluted sample is usually abovethe CMC in order to obtain a functional solubilisation of the antibody.In the products which were investigated here, the CMC of the detergentsindeed exceeded their CMC (polysorbate 80: 700 mg/l (50 fold excess))leading to the assumption that polysorbate 80 is present in form ofmicelles. In the above described protocol the concentration of thedetergent is reduced by dilution so that the concentration of thedetergent is near/drops below the CMC value (polysorbate 80: 14 mg/l or10.6 μM). Dilution of the detergent to near-CMC concentrations mayeliminate the micellar compartmentalization, and therefore, render theCSE molecules spiked accessible for the LAL enzymes.

Accordingly, the problem of the LER effect, (e.g. in the event sodiumcitrate and polysorbate 80 are used for the formulation of apharmaceutical product) can now be considered as being solved. Inconclusion, herewith provided is a safe, robust and reproducible testingmethod for pharmaceutical products.

In summary, in rituximab and rituximab placebo the above describedprotocol surprisingly overcomes the LER effect. By contrast, the sameprotocol could not reveal satisfactory results for NeoRecormon® (whichdoes not comprise an antibody but epoetin-beta) indicating that theherein provided methods are particularly useful for antibodyformulations, preferably for formulations with monoclonal antibodies,citrate buffer and polysorbate 80.

Example 2.2: Modified Protocol (1) for Overcoming the LER Effect

In this Example a modified protocol has been used which neverthelessovercomes the LER effect. The most important changes compared to Example2.1 are as follows:

-   1. In Example 2.2 the Spin Dialyzer has been used. In contrast, in    Example 2.1 the FastSpinDIALYZER is used which has more efficient    dialysis chambers and increases the efficiency of the dialysis (the    membranes are on both sides of the cylinder).-   2. In Example 2.2 the MWCO of the dialysis membrane is 500 Da. In    contrast, in Example 2.1 the MWCO of the dialysis membrane is 10    kDa.-   3. In Example 2.2 the dilution is 1:10 with endotoxin-free water. In    contrast, in Example 2.1 the dilution 1:10 with Tris-buffer pH˜7    (i.e. Tris/HCL buffer pH˜7). By diluting the sample with    endotoxin-free water at a ratio of 1:10 the pH value of the sample    is adjusted to about pH 6.0.-   4. In Example 2.2 the dialysis time is 4 h. In contrast, in Example    2.1 the dialysis time is 24 h.

In this Example rituximab and rituximab placebo were used as sample.However, for the same reasons as discussed with respect to Example 2.1,this protocol is useful for overcoming the LER in all typical antibodyformulations.

In particular, the protocol used in Example 2.2 is detailed as follows.

Protocol Overview

-   Step 1: “Setting up the LER effect” (see also below “LER positive    control”):    -   Rituximab and rituximab placebo samples were spiked with 5 EU/ml        or 0.5 EU/ml (CSE; Lonza, E. coli 0055:B5) and the mixture was        shaken for 60 min at room temperature at maximum speed [Shaker:        Heidolph Multi Reax, high speed (2,037 rpm)] to obtain a        “positive LER-effect” sample.-   Step 2: Adding MgCl₂: Before dialysis, add 2 M MgCl₂ stock solution    so that the final conc. is about 50 mM MgCl₂; 1 min shaking as in    step 1.-   Step 3: 1:10 Dilution [one sample without dilution (undiluted) as    reference]; shaking for 1 min as in step 1.-   Step 4: Dialysis for 4 h using a 500 Da membrane (30 min    pre-incubated with 0.2% BSA; optionally but not mandatory), exchange    of water after 2 h once. Transferring of the solution from the    dialysis chamber into a glass vial and shaking as in step 1 for 20    min at RT (room temperature, i.e. 21±2° C.).-   Step 5: kinetic LAL-assay measurement.

Detailed Protocol Step 1: Preparation of the Samples

-   -   Preparation antibody solution (rituximab) for 50 mM MgCl₂: Fill        1 tube with 877.5 μl rituximab+97.5 μl CSE (stock solution of        50 (5) EU CSE/ml→5 (0.5) EU/ml final concentration).    -   Unspiked control: 877.5 μl rituximab placebo+97.5 μl water    -   Unspiked water control: 975 μl water (for blank subtraction)    -   used vials: clear flat bottom small opening 1.5 ml Macherey &        Nagel, Ref. Nr. 70213    -   1 h shaking on Heidolph Multi Reax, high speed (2,037 rpm) at        room temperature.        Step 2: Addition of MgCl₂ to a Final Concentration of 50 mM        MgCl₂    -   Stock solution 1M MgCl₂.6H₂O: add 50 μl of a 1M MgCl₂-stock        solution to the spiked sample as well as to the unspiked sample        (blank).

Step 3: Dilution

-   -   Sample rituximab with 50 mM MgCl₂: prepare a 1:10 dilution by        adding 900 μl endotoxin-free water (i.e. LAL water)+100 μl        sample    -   Water control is treated the same with endotoxin-free water        (i.e. LAL water) instead of rituximab: dilute 1:10

Step 4: Dialysis

-   -   1 min shaking before dialysis [i.e. in a Heidolph Multi Reax        shaker, high speed (2,037 rpm) at room temperature].    -   Put samples into the 1 ml dialyzer chambers (Harvard Spin        Dialyzer) to which a membrane with MWCO 500 Da (optionally, 30        min pre-incubated with 0.2% BSA) is fixed.    -   Dialyze 4 h against 1 l Aqua Braun (i.e. sterile, pyrogen free        water; as supplied by B. Braun, Melsungen) at 24° C.; change        water after 2 h. Changing water has been tempered also to 24° C.    -   The Spin Dialyzers are distributed (depending on the number of        dialysis chambers) over multiple 21 beakers filled with 1 l Aqua        Braun under stirring (magnetic Teflon stirrer).    -   There are at most 5 Dialyzer in one 21 beaker.

Step 5: Preparation of the LER Positive Control

Also in this Example a LER positive control is used in the LAL assay.This LER positive control can be prepared at any time, provided that itis ready if the LAL assay starts. Advantageously, the LER positivecontrol is prepared 1 h before the end of 4 h dialysis, so that allsamples are ready for testing at the same time. For preparing the LERpositive control the following protocol is used:

-   -   rituximab 900 μl+100 μl CSE→final conc. CSE: 5.0 EU/ml.    -   Shaking in a Heidolph Multi Reax, high speed (2,037 rpm) at RT        for 1 h. Only under these conditions the max. LER effect (<1%        recovery rate) will be obtained.    -   In parallel prepare the following blanks:        -   Water with 5.0 EU/ml CSE        -   Water with 5.0 EU/ml CSE; diluted 1:10 (0.5 EU/ml).

Step 6: LAL Assay

-   -   Start test after all samples are prepared.    -   From all samples two aliquots of 100 μl are used for repeat        determination (2×, i.e. determination in duplicates) in a plate        which is incubated 10 min at 37° C. in the Tecan Reader.    -   Add 100 μl LAL Reagent (Kinetic-QCL™ Assay; Lonza) to each        sample in a well-defined sequence (according to the read-out of        the machine).

Results and Discussion:

The protocol described in Example 2.1 resulted in best reproduciblerecovery rates (also with respect to the water controls). However, theprotocol described in Example 2.2 resulted in a good CSE recovery-rateranging from 50 to 95% for both CSE concentrations spiked (see FIG. 4(B)[rituximab 046]). Therefore, it can be concluded that the protocol usedin Example 2.2 represents a functional equivalent to the protocoldescribed in Example 2.1.

Reference Example 1: Time Dependency of the LER Effect

In the prior art it is assumed that the LER effect appears immediatelyafter spiking of the sample with a defined amount of CSE (C. Platco,2014, “Low lipopolysaccharide recovery versus low endotoxin recovery incommon biological product matrices”. American Pharmaceutical Review,Sep. 1, 2014, pp. 1-6). Therefore, first the samples were shaked afterLPS spiking for a rather short time of about 2-10 min at roomtemperature. However, this kind of spiking turned out to be inefficientand some experiments indicated that the masking effect of the materialspiked has not yet reached its maximum during this short time interval(<10 min). It was found that the mechanism of spiking is one of thefundamental processes in analyzing the LER effect in a correct way (see,e.g., FIG. 13 [rituximab 027]). According to these data, the LER effectis a kinetic phenomenon, which requires time to mask the CSE moleculese.g. by penetrating into the micelles of the formulation mixture. Thus,shaking for 2-10 min prior to the next step for analyzing the LEReffect, as it represents the routine practice, are inappropriate andcannot be considered to be representative for the LER effect, becausethe conditions for its formation has not yet been reached. Therefore, aninternal standard to test the “positive LER effect” (defined to bepresent in case the recovery rate of 0% by the LAL test has beenreached) was included in the experiments. By performing a kinetic studyon the LER effect in rituximab, the positive LER effect was demonstratedto need ≥60 min incubation time.

In particular, it was analyzed how long shaking has to be carried out[max. frequency (i.e. vortexing) in a Heidolph Multi Reax shaker, highspeed (2,037 rpm) at room temperature in (21° C.±2° C.) a 1.5 ml clearglass, crimp neck, flat bottom vessel], in order to achieve the maximumLER effect. Therefore, rituximab samples were spiked with CSE in a vial,so as to obtain 0.5 and 5.0 EU/ml (vials by Macherey-Nagel, 1.5 ml).After spiking, the samples were shaked for 60 min, 30 min, 10 min, 5min, or 2 sec, respectively. Afterwards, 1:10 dilutions were prepared bymixing 900 μl endotoxin-free water (i.e. LAL water) with 100 μl sample.After dilution, the samples were again shaked for 1 min. Subsequently,the samples were tested in the LAL assay in duplicates. In particular,100 μl of each sample was applied onto a plate and incubated in thereader for 10 min at 37° C. Then, 100 μl chromogen was added to eachsample and the measurement was carried out. In this experiment, allsolutions had room temperature. As can be seen in FIG. 13 [rituximab027], the LER effect is lower (i.e. the recovery rate is higher) if thesample is diluted at a ratio of 1:10 as compared to the correspondingundiluted sample. In addition, after 2 sec shaking the recovery valuesfor the diluted samples with 5.0 EU/ml endotoxin were still atapproximately 50%. However, increasing shaking (i.e. vortexing) timeresults in a constant decrease of the recovery rate (with exception ofthe 30-min value), see FIG. 13 [rituximab 027]. In contrast, theundiluted samples show the maximum LER effect already after 2 sec.However, also the diluted samples showed a significant LER effect in thesamples which have been shaked (i.e. vortexed) for 60 min.

From this result it was concluded that spiking needs time to mask theLPS molecules into the detergent micelles. The “positive LER effect” iscomplete when about 100% masking or <0.5% recovery rates of CSE areobtained. This process requires a minimum of 1 h during shaking at roomtemperature [e.g., shaker: Heidolph Multi Reax, high speed (2,037 rpm)for 1 h at room temperature in a 1.5 to 5 ml clear glass, crimp neck,flat bottom] or alternatively storage at 4° C. for a longer timeperiod >24 h. The resulting “positive LER control” is shown in allgraphical plots as one bar in the graphical presentations at the rightside of the diagram.

Reference Example 2: Influence of Human Serum Albumin (HAS) andDifferent MgCl₂ Concentrations on the Recovery Rate

In order to determine the effect of HSA and different MgCl₂concentrations on the recovery rate of endotoxin spiked rituximabsamples, the following experiment has been performed. In addition, inthis experiment the influence of dialysis on the recovery rate has beenanalyzed. More specifically, rituximab spiked samples were shaken for 60min in order to obtain the “positive LER effect”. Prior to the dialysis,10-75 mM MgCl₂ were added, subsequently, a dilution was performed. NoBSA-blocked membrane was used. After the dialysis, 0.01 μg/ml HSA iseither added or not added. Subsequently, shaking for 20 min isperformed. In addition, some samples were not dialyzed at all. Inparticular, the different samples which have been tested in the LALassay are shown in FIG. 5 (i.e. [rituximab 059]). In this experiment,the LER effect could be overcome in some samples without dialysis.However, further experiments demonstrated that without dialysis the LEReffect cannot reproducibly been overcome. Or, in other words, withoutdialysis, the LER effect is sometimes overcome and sometimes not. Thus,dialyzing the samples results in a more robust method for overcoming theLER effect.

The samples have been prepared in a 1.5 ml screw neck vial byMacherey-Nagel.

Step 1: Preparation of the Samples

-   -   Preparation of spiked rituximab for 10 mM MgCl₂: 897 μl        rituximab+99.8 μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked rituximab for 50 mM MgCl₂: 889 μl        rituximab+98.8 μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked rituximab for 75 mM MgCl₂: 883 μl        rituximab+98.1 μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked water for 10 mM MgCl₂: 897 μl water+99.8        μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked water for 50 mM MgCl₂: 889 μl water+98.8        μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked water for 75 mM MgCl₂: 883 μl water+98.1        μl CSE so that 5.0 EU/ml are obtained    -   Shake for 60 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 2: Addition of MgCl₂

-   -   A 4 M MgCl₂ stock solution (i.e. 511.437 mg MgCl₂.6H2O in 0.629        ml water) was used.        -   For 10 mM MgCl₂ 2.5 μl of the 4 M solution are added to the            spiked sample.        -   For 50 mM MgCl₂ 12.5 μl of the 4 M solution are added to the            spiked sample.        -   For 75 mM MgCl₂ 19 μl of the 4 M solution are added to the            spiked sample.    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 3: Dilution

-   -   Dilutions at a ratio of 1:10 were prepared as follows:    -   Preparation rituximab 1:10: always 900 μl LAL water+100 μl        sample    -   The water was not diluted 1:10 since there are not enough        dialyzers available.    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 4: Dialysis

-   -   The samples were put into a 1 mL dialyzer. A 500 Da membrane.        However, the membrane was washed in LAL water.    -   Dialysis was performed against 1L Aqua Braun at 24° C. for 4 h,        and after 2 h the water was changed. The new water also had a        temperature of 24° C.    -   The dialyzers were located in three 2L beakers and rotated since        there was a long stirrer (i.e. stir bar) in each beaker.    -   There are always 4 dialyzers in each beaker.        Step 5: Addition of HSA after Dialysis    -   After the dialysis, the samples were portioned. For the        preparation of HSA-samples 396 μl of each sample were added to a        separate vial. For the preparation of samples without HSA 400 μl        were added to a separate vial.    -   To obtain a HSA concentration of 0.01 μg/ml, 4 μl of a 1 μg/ml        solution were added to the 396 μl samples    -   The HSA stock solution was newly prepared.    -   Shake for 20 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 6: Preparation of the LER Positive Control

-   -   The LER positive control is prepared 1 h before the end of the 4        h dialysis so that it is ready at the same time as the other        samples.    -   rituximab 900 μl+100 μl CSE of different CSE stock solutions so        that 5.0 EU/ml are obtained]        -   shaken at room temperature for 1 h [Heidolph Multi Reax            shaker, high speed (2,037 rpm)]

Step 7: LAL Assay

-   -   100 μl of each samples were applied onto a plate in double        determination    -   Incubation in the reader at 37° C. for 10 min.    -   100 μl chromogen were applied to each sample.    -   Starting the measurement in the reader.

Results and Discussion:

The results are shown in FIG. 5 [rituximab 059]. This experimentdemonstrates that HSA treatment reduces the recovery rate and istherefore less useful in context of the herein provided methods. Inaddition, the results show that BSA treatment of the dialysis membraneis not necessary to obtain satisfactory recovery rates. In addition,this experiment also demonstrates that 50 mM MgCl₂ is the optimum valuefor recovery, 10 and 75 mM MgCl₂ result in lower recovery. However, alsowith 75 mM MgCl₂ a satisfactory recovery rate was obtained. Moreover,this experiment shows that addition of MgCl₂ leads to a recovery withina satisfactory range (70-100%) even without dialysis. However, asmentioned above, without dialysis the LER effect cannot reproduciblybeen overcome. Thus, dialyzing the samples results in a more robustmethod for overcoming the LER effect. It is indicated that in theexperiment [rituximab 059] the water control values were high (part ofthe values >220%). The LER positive control is satisfactory; i.e. 0%recovery.

Reference Example 3: 4 Hours Incubation Time after Addition of MgCl₂

In this experiment rituximab samples were shaken for 60 min in order toachieve the “positive LER effect”. After addition of MgCl₂ the undilutedsamples were incubated for 4 h at room temperature). After thisincubation, the samples were shaked for 2 min. The different sampleswhich have been tested in the LAL assay are shown in FIG. 5(B)[rituximab 061].

-   -   The samples have been prepared in a 1.5 ml screw neck vial by        Macherey-Nagel.

Step 1: Preparation of the Samples

-   -   Preparation of spiked rituximab/water for 10 mM MgCl₂: 897 μl        rituximab/water+99.8 μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked rituximab/water for 50 mM MgCl₂: 889 μl        rituximab/water+98.8 μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked rituximab/water for 75 mM MgCl₂: 883 μl        rituximab/water+98.1 μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked rituximab/water for 100 mM MgCl₂: 877 μl        rituximab/water+97.5 μl CSE so that 5.0 EU/ml are obtained    -   Preparation of spiked rituximab/water for 150 mM MgCl₂: 866 μl        rituximab/water+96.3 μl CSE so that 5.0 EU/ml are obtained    -   Shake for 60 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 2: Addition of MgCl₂

-   -   A 4 M MgCl₂ stock solution (i.e. 534.661 mg MgCl₂. 6 H₂O in        0.657 ml water) was used.    -   For 10 mM MgCl₂ 2.5 μl of the 4M solution are added to the        spiked sample.    -   For 50 mM MgCl₂ 12.5 μl of the 4M solution are added to the        spiked sample.    -   For 75 mM MgCl₂ 19 μl of the 4M solution are added to the spiked        sample.    -   For 100 mM MgCl₂ 25 μl of the 4M solution are added to the        spiked sample.    -   For 150 mM MgCl₂ 37 μl of the 4M solution are added to the        spiked sample.    -   Shake for 1 min [high speed (2,037 rpm) at room temperature]

Step 3: Dilution

-   -   Dilutions at a ratio of 1:10 were prepared: 900 μl LAL water+100        μl sample (i.e. rituximab sample or water sample)    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 4: Preparation of the LER Positive Control

-   -   rituximab 900 μl+100 μl CSE so that 5.0 EU/ml are obtained    -   Another LER positive control was prepared by mixing 900 μl        rituximab+100 μl CSE so that 5.0 EU/ml are obtained; and        subsequently diluting the sample 1:10 with endotoxin-free water

Step 5: Shaking

-   -   All samples as well as the LER positive controls were shaken at        room temperature for 1 h [Heidolph Multi Reax shaker, high speed        (2,037 rpm)]

Step 6: LAL Assay

-   -   100 μl of each sample were applied onto a plate in double        determination    -   Incubation in the reader at 37° C. for 10 min.    -   100 μl chromogen was applied to each sample.    -   Starting the measurement in the reader.

Results and Discussion:

The result of this experiment is shown in FIG. 6(B) (i.e. [rituximab061]). This Figure demonstrates again that 50 mM MgCl₂ is thereproducible optimum value for CSE recovery; 10, 75 and 150 mM showslightly inferior results. When an incubation time after addition ofMgCl₂ was performed, the undiluted rituximab samples did not lead to aCSE recovery at all, see FIG. 6(B) (i.e. [rituximab 061]). However, the1:10 dilutions resulted in approximately 50-60% recovery (in particularwhen 10, 50, 75 or 100 mM MgCl₂ was added). The water control values aswell as the LER positive control were satisfactory. In this experimentno dialysis was performed. However, several experiments showed thatdialysis is necessary for reproducibly overcoming the LER effect.

Reference Example 4: Comparison of 2 and 4 Hours Incubation Time afterAddition of MgCl₂

The rituximab samples were shaken for 60 min in order to achieve the“positive LER effect”. After addition of MgCl₂ the undiluted sampleswere incubated for 2 or 4 h, then 1:10 diluted and measured in the LALassay. The different samples which have been tested in the LAL assay areshown in FIGS. 7(A) and 7(B) (i.e. [rituximab 062] and [rituximab 063],respectively).

-   -   The samples have been prepared in a 1.5 ml screw neck vial by        Macherey-Nagel.

Step 1: Preparation of the Samples

-   -   Preparation of rituximab/water for 10 mM MgCl₂: 897 μl        rituximab/water+99.8 μl of different CSE stock solutions so that        0.5 and 5.0 EU/ml are obtained.    -   Preparation of rituximab/water for 50 mM MgCl₂: 889 μl        rituximab/water+98.8 μl of different CSE stock solutions so that        0.5 and 5.0 EU/ml are obtained.    -   Preparation of rituximab/water for 75 mM MgCl₂: 883 μl        rituximab/water+98.1 μl of different CSE stock solutions so that        0.5 and 5.0 EU/ml are obtained.

Step 2: Preparation of Two LER Positive Controls

-   -   rituximab 900 μl+100 μl CSE so that 0.5 and 5.0 EU/ml are        obtained.    -   Dilution of one of the LER positive controls at a ratio of 1:10        with endotoxin-free water.

Step 3: Shaking

-   -   All samples as well as the LER positive control are shaken for 1        h: [Heidolph Multi Reax shaker, high speed (2,037 rpm) at room        temperature]

Step 4: Addition of MgCl₂

-   -   A 4 M MgCl₂ stock solution was used.        -   For 10 mM MgCl₂ 2.5 μl of the 4M solution are added to the            spiked sample        -   For 50 mM MgCl₂ 12.5 μl of the 4M solution are added to            spiked sample        -   For 75 mM MgCl₂ 19 μl of the 4M solution are added to spiked            sample    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 5: Incubation Time

-   -   The (undiluted) samples (as well as the LER positive controls)        are portioned. One half of each sample (about 500 μl) was        incubated for 2 h and the other half was incubated for 4 h,        respectively.

Step 6: Dilution

-   -   Shake for 2 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]    -   Dilutions at a ratio of 1:10 were prepared: 900 μl LAL water+100        μl sample (i.e. rituximab sample or water sample).    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 7: LAL Assay

-   -   100 μl of each samples were applied onto a plate in double        determination    -   Incubation in the reader at 37° C. for 10 min.    -   100 μl chromogen were applied to each sample.    -   Starting the measurement in the reader

Results and Discussion:

The results are shown in FIGS. 7(A) and 7(B) (i.e. [rituximab 062] and[rituximab 063], respectively). Here the recovery rates of 0.5 and 5.0EU/ml endotoxin was measured. When 10-75 mM MgCl₂ was added to thesamples, the recovery was the same in the samples which were incubatedfor 2 h (FIG. 7(A), i.e. [rituximab 062]) and in the samples which wereincubated for 4 h (FIG. 7(B), i.e. [rituximab 063]). In both experimentsthe recovery rates are very similar. In addition, in the samples whichwere spiked with 5.0 EU/ml endotoxin satisfactory recovery rates(80-90%) were obtained, even without dialysis. In the samples which werespiked with 0.5 EU/ml endotoxin the recovery rates were approximately35-45%. Importantly, without dilution (at a ratio of 1:10), complete LERis observed, i. e. 0% recovery, also in the presence of 10-75 mM MgCl₂.The water controls as well as the LER positive controls weresatisfactory. In these experiments no dialysis has been performed.However, further experiments demonstrated that without dialysis, the LEReffect is sometimes overcome and sometimes not. Thus, dialyzing thesamples results in a more robust method for overcoming the LER effect.

Reference Example 5: Comparison of 2 Hours Incubation Time afterAddition of Different Amounts of MgCl₂ with No Incubation Time afterAddition of Different Amounts of MgCl₂

The rituximab samples were shaken for 60 min in order to achieve the“positive LER effect”. After addition of MgCl₂ the undiluted sampleswere either not incubated or incubated for 2 h. Then 1:10 diluted andmeasured in the LAL assay. The different samples which have been testedin the LAL assay are shown in FIGS. 8(A) and 8(B) (i.e. [rituximab 064]and [rituximab 065], respectively).

Reference Example 5.1: No Incubation Time after Addition of MgCl₂

In this experiment, no incubation was performed after addition of MgCl₂to the samples.

The samples have been prepared in a 1.5 ml screw neck vial byMacherey-Nagel.

Step 1: Preparation of the Samples

-   -   Preparation of rituximab/water for 10 mM MgCl₂: 897 μl        rituximab/water+99.8 μl of different CSE stock solutions so that        0.5 and 5.0 EU/ml are obtained    -   Preparation of rituximab/water for 25 mM MgCl₂: 895 μl        rituximab/water+99.4 μl of different CSE stock solutions so that        0.5 and 5.0 EU/ml are obtained.    -   Preparation of rituximab/water for 50 mM MgCl₂: 889 μl        rituximab/water+98.8 μl of different CSE stock solutions so that        0.5 and 5.0 EU/ml are obtained.

Step 2: Preparation of Two LER Positive Controls

-   -   rituximab 900 μl+100 μl CSE so that 0.5 and 5.0 EU/ml are        obtained.    -   Dilution of one of the LER positive controls at a ratio of 1:10        with endotoxin-free water

Step 3: Shaking

-   -   All samples as well as the LER positive control were shaken        (i.e. vortexed) for 60 min [Heidolph Multi Reax shaker, high        speed (2,037 rpm) at room temperature]

Step 4: Addition of MgCl₂

-   -   A 4 M MgCl₂ stock solution was used.        -   For 10 mM MgCl₂ 2.5 μl of the 4M solution are added to the            spiked sample        -   For 25 mM MgCl₂ 6.25 μl of the 4M solution are added to            spiked sample        -   For 50 mM MgCl₂ 12.5 μl of the 4M solution are added to            spiked sample    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 5: Dilution

-   -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]    -   Dilutions at a ratio of 1:10 were prepared.    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 6: LAL Assay

-   -   100 μl of each samples were applied onto a plate in double        determination    -   Incubation in the reader at 37° C. for 10 min.    -   100 μl chromogen were applied to each sample.    -   Starting the measurement in the reader.

Results and Discussion:

The results are shown in FIG. 8(A) (i.e. [rituximab 064]). For thediscussion of the results see Reference Example 4.2.

Reference Example 5.2: Incubation of 2 h after Addition of MgCl₂

In this experiment, the samples were incubated for 2 h after addition ofMgCl₂. Steps 1 to 4 were performed as described above under ReferenceExample 4.1. However, after addition of MgCl₂ the undiluted samples wereincubated for 2 h at room temperature (21° C.)). After the incubation,the following steps 5 and 6 were performed. The different samples whichhave been tested in the LAL assay are shown in FIG. 8(B) (i.e.[rituximab 065]).

Step 5: Dilution

-   -   Shake for 2 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]    -   Dilutions at a ratio of 1:10 were prepared: 900 μl LAL water+100        μl sample (i.e. rituximab sample or water sample)    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 6: Preparation of Two LER Positive Controls

-   -   rituximab 900 μl+100 μl CSE so that 0.5 and 5.0 EU/ml are        obtained.    -   Dilution of one of the LER positive controls at a ratio of 1:10        with endotoxin-free water

Step 7: Shaking

-   -   All samples as well as the LER positive control were shaken for        1 h [Heidolph Multi Reax shaker, high speed (2,037 rpm) at room        temperature]

Step 8: LAL Assay

-   -   100 μl of each samples were applied onto a plate in double        determination    -   Incubation in the reader at 37° C. for 10 min.    -   100 μl chromogen were applied to each sample.    -   Starting the measurement in the reader.

Results and Discussion:

The results of Reference Example 4.1 are shown in FIG. 8(A) (i.e.[rituximab 064]); the results of Reference Example 4.2 are shown in FIG.8(B) (i.e. [rituximab 065]). In these two experiments, dilution and LALmeasurement was either carried out immediately after addition of MgCl₂(FIG. 8(A), [rituximab 064]) or after leaving the sample to rest for 2 hafter addition of MgCl₂ (FIG. 8(B), [rituximab 065]). All recoveryvalues were very similar and in the samples which were spiked with 5.0EU/ml CSE, satisfactory (60-80%) recovery rates have been obtained evenwithout dialysis. Interestingly, after an incubation time of 2 h, the 25mM MgCl₂ concentration resulted in 100% recovery. Thus, an incubationtime after addition of MgCl₂ seems to be a valuable measure to decreasethe LER effect. However, the recovery values for the samples which werespiked with 0.5 EU/ml CSE were low with approximately 20-35%. Thisindicates that beside addition of Mg²⁺ and dilution, dialysis representsa necessary step for reliably overcoming the LER effect. In theseexperiments the water control values were satisfactory. The undilutedLER positive control was also satisfactory, i.e. 0%.

Reference Example 6: Comparison of Different Dilutions with Rituximaband Rituximab Placebo Samples

After spiking, rituximab and rituximab placebo samples were shaken for60 min in order to achieve the “positive LER effect”. After addition ofMgCl₂ the undiluted samples were shaked for 1 h and diluted at a ratioof 1:2, 1:5, 1:10 or 1:20 Afterwards the LAL assay was performed. Thedifferent samples which have been tested in the LAL assay are shown inFIG. 9 (i.e. [rituximab 072]).

In particular, the following experiment has been performed:

Step 1: Preparation of the Samples

-   -   Preparation of rituximab/rituximab placebo/water for 25 mM        MgCl₂: 895 μl rituximab/rituximab placebo/water+99.4 μl of        different CSE stock solutions so that 0.5 and 5.0 EU/ml are        obtained.

Step 2: Preparation of Three LER Positive Controls

-   -   rituximab placebo 450 μl+50 μl CSE so that 0.5 und 5.0 EU/ml are        obtained (first LER positive control).    -   rituximab 450 μl+50 μl CSE so that 0.5 und 5.0 EU/ml are        obtained (second LER positive control).    -   rituximab 450 μl+50 μl CSE so that 0.5 und 5.0 EU/ml are        obtained. Afterwards, this sample was diluted at a ratio of 1:10        (third LER positive control).

Step 3: Shaking

-   -   All samples as well as the LER positive controls were shaken        (i.e. vortexed) for 60 min [Heidolph Multi Reax shaker, high        speed (2,037 rpm) at room temperature]

Step 4: Addition of MgCl₂

-   -   A 4 M MgCl₂ stock solution was used.        -   For 25 mM MgCl₂ 6.25 μl of the 4M solution are added to            spiked sample        -   Shake for 1 min [Heidolph Multi Reax shaker, high speed            (2,037 rpm) at room temperature]

Step 5: Dilution

-   -   The samples were diluted as follows:        -   Dilution at a ratio of 1:5: 400 μl water+100 μl sample        -   Dilution at a ratio of 1:10: 450 μl water+50 μl sample        -   Dilution at a ratio of 1:20: 475 μl water+25 μl sample    -   One of the three LER positive controls was diluted at a ratio of        1:10.    -   The water without MgCl₂ was not diluted.    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 6: LAL Assay

-   -   100 μl of each samples were applied onto a plate in double        determination    -   Incubation in the reader at 37° C. for 10 min.    -   100 μl chromogen was applied to each sample.    -   Starting the measurement in the reader.

Results and Discussion:

The results are shown in FIG. 9 (i.e. [rituximab 072]). Importantly, theresults for rituximab and rituximab placebo show no significantdifferences. This indicates that the LER effect in rituximab is mainlybased on the buffer system (i.e. citrate buffer with polysorbate 80) andthat the antibody (i.e. rituximab) does not have a significant impact onthe LER effect. However, in this experiment the recovery rates areunsatisfactory for both rituximab and rituximab placebo. In theexperiment described above, the water control values were satisfactory.The undiluted LER positive controls were satisfactory, too, withrecovery rates of 0%.

Reference Example 7: Influence of Incubation Time Before Addition ofMgCl₂ on the Recovery Rate in Rituximab and Rituximab Placebo Samples

In the following experiment it was tested whether incubation timesbefore addition of MgCl₂ have an influence on the recovery rate ofrituximab and rituximab placebo samples. In particular, rituximab andrituximab placebo samples were shaken for 60 min in order to achieve the“positive LER effect”. Then the samples were incubated at 4° C. for 0 hto 3 days. Afterwards, MgCl₂ was added to a concentration of 50 mM andthe samples were diluted. Then, dialysis was performed with a dialysismembrane which was not BSA-blocked. The different samples which havebeen tested in the LAL assay are shown in FIG. 10 (i.e. [rituximab 079],and [rituximab 082]).

The samples have been prepared in a 1.5 ml screw neck vial byMacherey-Nagel.

Step 1: Preparation of the Samples

-   -   Preparation rituximab/rituximab placebo/water for 50 mM MgCl₂:        5,346 μl rituximab/rituximab placebo/water+596 μl of different        CSE stock solutions so that 0.5 or 5.0 EU/ml were obtained.    -   Vortex for 60 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]    -   For the further procedure 1 ml of each sample was transferred        into new vials

Step 2: Incubation Time

-   -   The samples were put into the refrigerator at 4° C. for 0 h, 4        h, 1 day, 3 days, or 7 days.    -   After the incubation time of 1 day, 3 days or 7 days the samples        were shaked for 2 min [Heidolph Multi Reax shaker, high speed        (2,037 rpm) at room temperature]. After 0 h and 4 h incubation        time the samples were not shaked.

Step 3: Addition of MgCl₂

-   -   A 5 M MgCl₂ stock solution (i.e. 0.9055 g MgCl₂ in 0.891 ml        water) was used        -   For 50 mM MgCl₂10 μl of the 5 M solution were added to the            spiked samples    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature

Step 4: Dilution

-   -   Dilutions at a ratio of 1:10 were prepared: 900 μl LAL water+100        μl sample (i.e. rituximab sample, rituximab placebo sample or        water sample)    -   The water without MgCl₂ was not diluted.    -   Shake for 1 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature

Step 5: Dialysis

-   -   The samples were added into a 1 ml dialyzer. Dialysis was        performed against 1 l Aqua Braun at 24° C. for 4 h, and after 2        h the water was changed. The new water also had a temperature of        24° C. A 500 Da membrane was used which has not been incubated        with BSA before.    -   The dialyzers were located in three 2 l beakers and rotated        since there was a long stirrer (i.e. stir bar) in each beaker.    -   After dialysis the samples have been transferred into new 1.5 ml        vials.

Step 6: Shaking

-   -   Shake for 20 min [Heidolph Multi Reax shaker, high speed (2,037        rpm) at room temperature]

Step 7: Preparation of the LER Positive Control

-   -   The LER control was prepared 1 h before the end of the 4 h        dialysis so that it is ready at the same time as the other        samples.    -   rituximab or rituximab placebo 900 μl+100 μl CSE to obtain 5.0        EU/mL CSE    -   Shake at room temperature for 1 h [Heidolph Multi Reax shaker,        high speed (2,037 rpm)]

Step 8: LAL Assay

-   -   100 μl of each samples were applied onto a plate in double        determination    -   Incubation in the reader at 37° C. for 10 min.    -   100 μl chromogen were applied to each sample.    -   Starting the measurement in the reader.

Results and Discussion:

The results are shown in FIG. 10(A) (i.e. [rituximab 079], noincubation), FIG. 10(B) (i.e. [rituximab 080], 4 h incubation), FIG.10(C) (i.e. [rituximab 081], 1 day incubation), and FIG. 10(D) (i.e.[rituximab 082], 3 days incubation). The results for the 7 daysincubation are not shown. The results again demonstrate that by usingthe herein described protocols good recovery rates can be obtained forrituximab as well as for rituximab placebo samples. In addition, thisexperiment demonstrates that an incubation time before addition of MgCl₂does not improve the recovery rates. In particular, while the incubationtime of 0-4 h led to recovery rates which are within the desired range(50-200%), the incubation times of 1 day and 3 days led to lowerrecovery rates (approximately 20-30%). If no (i.e. 0 h) incubation wasperformed, very good recovery rates were obtained for rituximab(70-80%). Also for rituximab placebo which was spiked with 5.0 EU/mlCSE, the recovery rate was satisfactory. The recovery rate for rituximabplacebo which was spiked with 0.5 EU/ml CSE was negative since the blankwas very high. This may indicate that this blank sample was contaminatedwith endotoxin. In this experiment also recovery rates of the watercontrol (80-120%) as well as of the LER positive control (0%) weresatisfactory.

Reference Example 8: The Protocol of the State of the Art (“LAL Assay”)and Modifications Thereof Cannot Overcome the LER Effect in Rituximab orRituximab Placebo

In this Example it was determined whether the commonly known LAL assayis able to detect endotoxins in rituximab and rituximab placebopreparations. Therefore, the following materials have been used:

[rituximab 002]: Lonza CSE+Lonza reagent (i.e. Lonza kit)[rituximab 004]: ACC CSE or Lonza CSE, respectively+ACC reagent (i.e.ACC kit)[rituximab 005]: Lonza CSE+ACC reagent (i.e. ACC kit)

The LAL assays have been precisely been performed as described by themanufacturer.

As can be seen from FIGS. 11(A)-(C) (i.e. [rituximab 002], [rituximab004] and [rituximab 005], respectively), the standard LAL assay did notlead to satisfactory recovery rates, even if several different dilutionsare tested. In particular, in one experiment (i.e. [rituximab 002]),rituximab was pipetted into a 96-well plate (i.e. a microtiter plate)and spiked with Lonza CSE to a final concentration of 0.5 EU/ml or 5.0EU/ml. Subsequently, dilutions with water as shown in FIG. 11(A) (i.e.[rituximab 002]) were carried out in the 96-well plate. Then a LAL assaywas performed. However, as can be seen in FIG. 11(A) (i.e. [rituximab002]), 50% recovery was not reached.

In a similar experiment (i.e. [rituximab 004]) rituximab was pipettedinto the wells of a microtiter plate and spiked with Lonza CSE and ACCCSE to a final concentration of 0.5 EU/ml or 5.0 EU/ml. Subsequently,dilutions with water as shown in FIG. 11(B) (i.e. [rituximab 004]) werecarried out in the 96-well plate. Afterwards, measurement was performed.However, as can be seen in FIG. 11(B), with ACC CSE a recovery which ismore than 200% was obtained and also the Lonza CSE spiking did notresult to satisfactory recovery rates.

In further experiments, the effect of pH adjustment on the LAL assay wasanalyzed. In particular, in one experiment [rituximab 005] rituximab waspipetted into a microtiter plate and Lonza CSE spiking was performed inthe plate. Subsequently, the dilutions with water or the pH adjustmentas indicated in FIG. 11(C) [rituximab 005] were performed. However,neither the dilution nor the pH adjustment resulted in a recovery of 50%(see FIG. 11(C)[rituximab 005]).

Also dialysis alone does not result in a satisfactory recovery rate.More specifically, in a further experiment, rituximab was spiked withCSE to result in a final concentration of 0.5 and 5.0 EU/ml (i.e. 900 μlrituximab solution was mixed with 100 μl CSE). Subsequently, the sampleswere dialysed in a 1 ml Spin Dialyser (in 1 ml Teflon chambers) for 4hours at 4° C. with one change of water after 2 h. The dialysis membranehad a MWCO of 100 Da. Then, dilutions as shown in FIG. 12 (i.e.[rituximab 011]) were performed in the plate. Subsequently, endotoxinrecovery was measured by using the LAL assay. However, good recoveryrates could only be obtained for the water controls. In the case ofrituximab the maximum recovery was <5% (see FIG. 12, [rituximab 011]).Thus, only dialysis and dilution does not overcome the LER effect.

Reference Example 9: Hold Time Studies

To identify and monitor the LER effect, endotoxin contents have beenmonitored over time in an endotoxin hold time study. Therefore, anundiluted sample of various buffers has been spiked with endotoxin andstored over time (up to 28 days). Acceptable endotoxin values recoveredin the PPC after spiking with the appropriate sample mixture are definedto be in the range of 50-200% of the theoretical spike value (100%). TheLER effect is indicated by a significant loss of endotoxins over time.In particular, an adverse trend of endotoxin values <50% of thetheoretical spike value are indicative for the LER effect.

Several formulation buffer components were studied in an endotoxin holdtime study (for results see the following table).

TABLE 1 Hold time studies endotoxin endotoxin recovery [EU/ml] at timespike start excipient [EU/ml] (T₀) day 7 day 14 day 21 day 28α,α-trehalose 5 4.85 4.65 3.48 4.32 4.51 NaH₂PO₄ 5 5.27 4.64 3.3  3.4 3.37 Na₂HPO₄ 5 5.99 5.72 5.07 5.63 5.06 Polysorbate 20 5 4.04 4.03 4.233.94 4.16 Polysorbate 20 + 5 0.13 0.18 n.d. n.d. n.d. Na₂HPO₄Polysorbate 20 + 5 0.37 0.77 n.d. n.d. n.d. NaH₂PO₄ Na₂HPO₄ + 5 5.124.64 n.d. n.d. n.d. NaH₂PO₄ Polysorbate 20 + 5 0.91 <0.1 n.d. n.d. n.d.Na₂HPO₄ + NaH₂PO₄ sodium citrate- 5 5.2 4.7 4   3.72 4.28 dihydratePolysorbate 80 5 3.25 3.15 3.27 3.05 3.17 NaCl 5 5.76 5.72 5.45 4.746.49 Na citrate + 5 1.33 0.16 n.d. n.d. n.d. polysorbate 80 + NaCl Nacitrate + 5 0.8 <0.1 n.d. n.d. n.d. polysorbate 80 polysorbate 80 + 52.94 2.45 n.d. n.d. n.d. NaCl Urea 5 5.14 6.21 5.66 5.28 5.29 L-Leu 55.72 5.61 5.87 5.01 5.88 L-Ile 5 5.70 5.82 6.24 5.17 6.17 L-Thr 5 5.545.65 5.66 4.76 5.88 L-Glu 5 5.28 5.03 5.50 4.43 4.24 L-Phe 5 5.49 5.505.96 4.98 6.34 Gly 5 4.64 4.57 4.75 4.27 5.00 n.d. = not determined

As can be seen from the above table, the buffers comprising polysorbate20 and Na₂HPO₄; polysorbate 20 and NaH₂PO₄; Polysorbate 20, Na₂HPO₄ ⁺and NaH₂PO₄; Na citrate, polysorbate 80 and NaCl; Na citrate andpolysorbate 80; as well as polysorbate 80 and NaCl exhibit a LER effect.

Reference Example 10: Influence of Buffer and Detergent on the LEREffect

In several experiments the effect of citrate and/or polysorbate 80 onthe LER effect was analyzed. In particular, in one experiment rituximaband 25 mM sodium citrate buffer were used as samples. Before spiking,the pH was adjusted to pH 7. Subsequently, CSE spiking was performed inthe plate, and the samples were diluted with water. As can be seen fromFIG. 14(A) (i.e. [rituximab 006]), a satisfactory recovery rate could beobtained for sodium citrate by using a dilution of 1:10. However, in thecase of rituximab a recovery of 50% could not be reached.

In another experiment 25 mM sodium citrate buffer, polysorbate 80 and acombination of both were used as samples. In particular, theconcentrations as present in Rituximab were used (i.e. polysorbate 80:0.7 mg/ml; sodium citrate: 9 mg/ml). These buffer systems were spikedwith 0.5 and 5.0 EU/ml of Lonza CSE or with Cape cod CSE (except ofsodium citrate, which was spiked with Lonza only, as ACC spiking ofsodium citrate buffer was already performed in experiment describedabove and shown in FIG. 14(A) (i.e. [rituximab 006]). After spiking, a1:2 or 1:5 dilution with water was performed in the plate. In the caseof polysorbate 80, several samples led to a satisfactory recovery ratebetween 50% and 200%. In contrast, in the case of sodium citrate buffer,only the 1:10 dilution led to a recovery rate between 50% and 200%. Thisindicates that the citrate buffer has a more significant impact on theLER effect as compared to polysorbate 80. Moreover, the LER effect couldnot be overcome in this experiment if a combination of sodium citrateand polysorbate 80 was used as a sample. This experiment indicates thatin monoclonal antibody formulations the LER effect is caused by thebuffer formulation (i.e. by the combination of sodium citrate buffer andpolysorbate 80.

These results have been verified by another experiment wherein severaldifferent dilutions were tested. In particular, samples comprisingeither 25 mM sodium citrate buffer (pH 6.5), 700 mg/L polysorbate 80 orboth (i.e. the formulation of rituximab) were prepared. Thesepreparations as well as water controls were spiked with Lonza CSE to afinal concentration of 0.5 or 5.0 EU/ml. All samples were shaken for 1hour at room temperature in the vortex machine [shaker: Heidolph MultiReax, high speed (2,037 rpm) in a 1.5 clear glass, crimp neck, flatbottom vessel Subsequently, the dilutions as indicated in FIG. 14(B)(i.e. [rituximab 029]) were performed with endotoxin-free water in 1.5ml vials and are shaken (as before) for 1 min. After shaking, the LALassay was performed. In particular, 100 μl of each of the samples wereadded to a 96-well plate and incubated in the reader for 10 min at 37°C. Then, 100 μl chromogen was added to each of the samples and themeasurement was carried out. As can be seen in FIG. 14(B) (i.e.[rituximab 029]) the LER effect of the buffer (i.e. the sodium citratebuffer) is stronger as compared to the LER effect of the detergent (i.e.polysorbate 80). While polysorbate 80 shows a relatively constantrecovery rate (˜40-90%, see FIG. 14(B), [rituximab 029], columns 2-5from the right), in citrate-buffer the LER effect is dependent on thedilution. Most importantly, a strong and reproducible LER effect isexpressed if polysorbate 80 is combined with citrate buffer (as it isthe case in monoclonal antibody formulations), see FIG. 14(B) (i.e.[rituximab 029]).

In these samples, also with high dilution, the recovery rate is only5-10%. Accordingly, this experiment demonstrates how a positive LEReffect can be obtained. This result is pioneering in the field ofendotoxin determination, as it allows for testing of several means andmethods for their ability to overcome the LER effect.

When analyzing the effect of buffer and detergent separately, the effectof the buffer on the LER effect was more pronounced in both NeoRecormon®and Rituximab (see, e.g. FIG. 14(B), i.e. [rituximab 029]). These datasurprisingly indicate that the removal of the buffer is more criticalthan removal of the detergent. Taking into account that theconcentrations of buffers in both formulations is comparable (rituximab:25 mM sodium citrate and NeoRecormon: 27.8 mM for sodium phosphate), thereason for this effect has to be found in the structure of the bufferand/or its physico-chemical properties. Sodium citrate is a well-knownchelating anion, whereas in phosphate this effect is less pronounced.Therefore, these observations may also explain why the addition of Mg²⁺is important for overcoming the LER effect, since Mg²⁺ is complexed bythe chelating buffer reducing its concentration in the LAL test.

Reference Example 11: Standard Physical and Biochemical Methods do notRecover Endotoxins Masked by the LER Effect

In order to overcome the LER effect (in the buffers identified as havingthe LER effect in Reference Example 9), different physical andbiochemical methods were tested:

Freezing of endotoxin spiked samples at −30° C. This study is based onthe initial finding that LER is more pronounced at room temperature ascompared to 2-8° C. Result: freezing of endotoxin spiked samples doesnot overcome LER.

Heating of endotoxin spiked samples for 30 minutes at 70° C. This studywas conducted because heating has shown to overcome endotoxin maskingeffects for some products (Dawson, 2005, LAL update. 22:1-6). Result:Heat treatment of endotoxin spiked samples does not overcome LER.

Dilution of endotoxin spiked samples to maximum valid dilution (MVD).This study was conducted since sample dilution is the standard method toovercome LAL inhibition. Result: As can be seen from FIG. 11 (i.e.[rituximab 002], [rituximab 004], [rituximab 005]), dilution alone doesnot overcome the LER effect.

Use of Endo Trap Columns for endotoxin spiked samples. These columnsserve to remove endotoxins from solutions via affinity chromatography. Atest was carried out with an aqueous endotoxin solution. Result:Endotoxins could not be recovered from the column.

Reference Example 12: Removal of Detergents by Dialysis

Several dialysis chambers and membranes (including different sizes ofthe molecular weight cut-off, MWCO) available on the market have beentested as detailed below.

Suitable membranes for dialysis chambers are commercially available are,e.g., cellulose acetate (MWCO 100 to 300,000 Da), regenerated cellulose(MWCO 1,000 to 50,000 Da), or cellulose ester (MWCO of 100 to 500 Da).Herein cellulose acetate and cellulose ester are preferred, celluloseacetate is most preferred.

The test samples (i.e. rituximab) were diluted prior to the dialysis,this way approaching the CMC and creating increasing levels of monomersof the detergent which were expected to diffuse through the dialysismembrane. Investigations on the recovery rate of the CSE spiked revealedthat in case of rituximab the regenerated cellulose was not so efficientas compared to the cellulose acetate. In a series of experiments withrituximab it was identified that the MWCO is preferably ˜10 kDa. Thissize is preferred because this size is thought to i) speed up thedialysis process and ii) allow also higher oligomeric aggregates (butnot micelles) of the detergent to pass through the membrane, in case thehydrophobic character of the cellulose acetate (acetyl esters on theglucose polymers) will not inhibit such kind of transportation process.

The experiment to determine the optimum for dialysis has been performedto mimic the situation in NeoRecormon®. As outlined earlier, buffer anddetergent were those compounds in the sample formulation which mostlyinfluenced the LER effect. In order to mimic the formulation ofNeoRecormon® a defined amount of phosphate buffer in a total volume of0.5 ml (2.7 mg) in the presence of 0.1 mg/ml poysorbate 20 was preparedand subjected to dialysis (in a spin dialyzer). In FIG. 1 is shown avery simple example of such experiment using a dialysis membrane ofcellulose acetate with a MWCO of 12-16 kDa. Here the material remainingin the inner dialysate after a given time is shown, this waydemonstrating the efficiency of the dialysis. In particular, the weightof material remaining in the inner dialysis chamber over a period of 72h (3d) was analyzed. The result is rather surprising as it shows thatcomplete and effective dialysis of NeoRecormon® is only achieved after alonger dialysis period at room temperature (>24-48 h). Based on thisexperiment the preferred dialysis time is 20 h to overnight (e.g. 24 h).In addition, this result indicates that Harvard Fast Dialyzer ispreferred over the Harvard Spin Dialyzer, the former having the doublearea of dialysis membrane, and thus leads to a quicker dialysis.

In FIG. 2, there is shown the efficiency of dialysis in case phosphateis placed into the inner dialysate compartment of the dialysis. In thisexperiment, the dialysis membrane (MWCO 12-16 kDa) was washed with 0.2%BSA (30 min) prior to its use, in order to avoid unspecific absorbanceof the CSE spiked to the sample. However, in the herein providedinventive methods a dilution (e.g. a dilution at a ratio of 1:10) of thesamples reduces the concentration of the detergent.

Reference Example 13: Influence of MgCl₂ on the LER Effect

It has been found that the LER effect could be reduced by addition ofMgCl₂ to the sample (see, e.g., FIG. 15 (i.e. [rituximab 031]). Inparticular, best results were observed when the concentration of Mg²⁺was twice the concentration of the sodium citrate (i.e. 50 mM Mg²⁺).

In particular, samples comprising either 25 mM sodium citrate buffer, pH6.5 (i.e. sodium citrate buffer, pH 6.5), 0.7 mg/ml polysorbate 80, orboth (with pH 6.5, i.e. the formulation of rituximab) were prepared.These preparations as well as water controls were spiked with Lonza CSEto a final concentration of 0.5 or 5.0 EU/ml. All samples were shakenfor 1 hour at room temperature [shaker: Heidolph Multi Reax, high speed(2,037 rpm) in a 1.5 clear glass, crimp neck, flat bottom vessel]. Then,MgCl₂ to reach a concentration of 10 mM, 25 mM, 50 mM or 75 mM was addedto the samples. Subsequently, the dilutions as indicated in FIGS. 15(A),18(B), 18(C), and 18(D) (i.e. [rituximab 030-rituximab 033]) wereperformed with endotoxin-free water in a 1.5 ml vial and shaken (i.e.vortexed as before) for 1 min. After shaking, the LAL assay wasperformed. In particular, 100 μl of each of the samples was added in a96-well plate and incubated in the reader for 10 min at 37° C.Afterwards, 100 μl chromogen was added to each of the samples and themeasurement was carried out. As can be seen in FIG. 15(A) (i.e.[rituximab 030]), in all diluted samples, MgCl₂ (10 mM) can neutralizethe complexing effect of citrate. Magnesium ions reduce the LER effectin samples which comprise polysorbate 80 as well as citrate buffer. Inthis case, a recovery of approximately 50% with 5.0 EU/ml and 25% with0.5 EU/ml was achieved. The control values of water range around thetheoretically expected value (i.e. 70-130%). Moreover, a comparison ofFIGS. 15(A), 15(B), 15(C), and 15(D) (i.e. [rituximab 030], [rituximab031], [rituximab 032] and [rituximab 033]) shows that a MgCl₂concentration which is twice the concentration of the citrate buffer(i.e. 50 mM MgCl₂) leads to best recovery rates. Although 25 mM and 75mM MgCl₂ are not the optimal concentration of MgCl₂, theseconcentrations nevertheless overcome the LER of the citrate buffer(recovery: 75-190%). In a similar experiment wherein rituximab was usedas a sample, it was demonstrated that only the addition of MgCl₂ to aconcentration of 10 mM, 50 mM, or 75 mM MgCl₂ and a subsequent dilutionat a ratio of 1:10 (without dialysis) was able to lead to a satisfactoryrecovery of endotoxin which was spiked to a final concentration of 5.0EU/ml (see FIG. 5, i.e. [rituximab 059]).

Reference Example 14: Effects of Mechanical Treatments on the LER Effect

It was tested whether mechanical treatments (such as shaking andultrasonification) are useful for dispersing the micelles, and thus forreducing the LER effect.

In particular, endotoxin-free water (i.e. LAL water) and rituximab werespiked with Lonza CSE to achieve a final concentration of 0.5 and 5.0EU/ml. Then, the samples were either sonicated for 1 hour or shaked for1 hour [i.e. vortexed in the Heidolph Multi Reax shaker at high speed(2,037 rpm) at room temperature in a 1.5 ml clear glass, crimp neck,flat bottom vessels]. Then 1:10 (sample:water) dilutions were preparedwith endotoxin-free water. Subsequently, the diluted samples weredialyzed by using a 12-16 kD membrane (which, before dialysis, had beenincubated in 0.2% BSA for 30 min). The dialysis took place in two 2 lbeakers for 4 hours. The external dialysate was 1 l Aqua Braun and thewater was changed after 2 hours of dialysis. After dialysis MgCl₂ wasadded to some of the samples (as indicated in FIG. 16, i.e. [rituximab034]) so as to result in a final concentration of MgCl₂ of 50 mM. Afteraddition of MgCl₂, all samples (also the samples without MgCl₂) wereshaken for 20 min (e.g. vortexed as before). Subsequently, the LAL assaywas performed. In particular, 100 μl of each of the samples was added toa 96-well plate and incubated in the reader for 10 min at 37° C. Then,100 μl chromogen was added to each of the samples and the measurementwas carried out. As can be seen in FIG. 16 (i.e. [rituximab 034], therecovery rates are neither improved by shaking nor by ultrasound.Therefore, it can be concluded that mechanically dispersing the LERcausing micelles by shaking or ultrasonification is compared theretoinefficient. However, addition MgCl₂ (50 mM) reduced the LER effect asit resulted in improved recovery values of up to 5-20%. In addition,this experiment also demonstrates that the order of the differentperformed steps is important for overcoming the LER effect. Inparticular, in the experiment described above (and shown in FIG. 16,i.e. [rituximab 034]), the order of the steps was (a) dilution, (b)dialysis, and (c) addition of MgCl₂. This order did not result insatisfactory recovery rates (see FIG. 16, i.e. [rituximab 034]).However, as demonstrated in FIGS. 3 and 4 (i.e. [rituximab 046],[rituximab 115] and [rituximab 117]), the order (a) addition of MgCl₂,(b) dilution, and (c) dialysis results in recovery rates which fulfillthe requirements of the FDA (i.e. 50%-200%).

The present invention refers to the following nucleotide and amino acidsequences:

SEQ ID NO: 1: Rituximab heavy chain, amino acid sequenceQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KSEQ ID NO: 2: Rituximab light chain, amino acid sequenceQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

1. A method for preparation of a sample, wherein the sample comprises an antibody, for a limulus amoebocyte lysate (LAL) assay, wherein the method comprises the following steps in the following order: (a) adding magnesium ions, preferably in form of MgCl₂, to the sample, (b) diluting the sample, and (c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution.
 2. A method for determining bacterial endotoxin in a sample comprising an antibody exhibiting the LER effect, wherein the method comprises the following steps in the following order: (a) adding magnesium ions, preferably in form of MgCl₂, to the sample, (b) diluting the sample, (c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution, and (d) determining bacterial endotoxin in the sample by using a LAL assay.
 3. The method of claim 1, wherein the antibody is a therapeutic antibody.
 4. The method of claim 1, wherein the antibody is formulated with polysorbate
 80. 5. The method of claim 1, wherein the antibody is formulated with a citrate buffer.
 6. The method of claim 1, wherein the antibody is formulated with an about 25 mM sodium citrate buffer and about 700 mg/polysorbate 80 and has a pH value of about 6.5.
 7. The method of claim 1, wherein the antibody is the anti-CD20 antibody rituximab.
 8. The method of claim 1, wherein in step (a) magnesium ions are added to a final concentration of about 25 to 75 mM.
 9. The method of claim 1, wherein in step (b) the pH-value of the sample is adjusted by diluting the sample with 10-50 mM Tris/HCl buffer pH 6.0-9.0, preferably 6.0-8.0.
 10. The method of claim 1, wherein in step (b) the sample is diluted at a ratio of 1:10.
 11. The method of claim 1, wherein during dialysis in step (c) the sample has a pH-value of 6.0-8.0.
 12. The method of claim 1, wherein in step (c) the dialysis takes about 24 hours at room temperature.
 13. The method of claim 1, wherein for the dialysis in step (c) a membrane with a molecular-weight cut-off of 10 kDa is used.
 14. The method of claim 1, wherein for the dialysis in step (c) a cellulose acetate membrane is used.
 15. The method of claim 1, wherein for the dialysis in step (c) the water is changed twice.
 16. The method of claim 1, further comprising the step of producing a low endotoxin recovery (LER) positive control by spiking a known amount of endotoxin into an aliquot of the sample and shaking the endotoxin spiked aliquot of the sample for 60 min to 2 hours.
 17. A method for rendering a sample comprising an antibody reactive to factor C in a LAL enzymatic cascade, comprising the steps of: (a) adding magnesium ions, preferably in form of MgCl₂, to the sample, (b) diluting the sample, and (c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution.
 18. The method of claim 2, wherein the antibody is a therapeutic antibody.
 19. The method of claim 2, wherein the antibody is formulated with polysorbate
 80. 20. The method of claim 2, wherein the antibody is formulated with a citrate buffer.
 21. The method of claim 2, wherein the antibody is formulated with an about 25 mM sodium citrate buffer and about 700 mg/I polysorbate 80 and has a pH value of about 6.5.
 22. The method of claim 2, wherein the antibody is the anti-CD20 antibody rituximab.
 23. The method of claim 2, wherein in step (a) magnesium ions are added to a final concentration of about 25 to 75 mM.
 24. The method of claim 2, wherein in step (b) the pH-value of the sample is adjusted by diluting the sample with 10-50 mM Tris/HCl buffer pH 6.0-9.0, preferably 6.0-8.0.
 25. The method of claim 2, wherein in step (b) the sample is diluted at a ratio of 1:10.
 26. The method of claim 2, wherein during dialysis in step (c) the sample has a pH-value of 6.0-8.0.
 27. The method of claim 2, wherein in step (c) the dialysis takes about 24 hours at room temperature.
 28. The method of claim 2, wherein for the dialysis in step (c) a membrane with a molecular-weight cut-off of 10 kDa is used.
 29. The method of claim 2, wherein for the dialysis in step (c) a cellulose acetate membrane is used.
 30. The method of claim 2, wherein for the dialysis in step (c) the water is changed twice.
 31. A method for rendering a sample comprising an antibody reactive to factor C in a LAL enzymatic cascade, comprising the steps of: (a) adding magnesium ions, preferably in form of MgCl₂, to the sample, (b) diluting the sample, (c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution, and (d) determining bacterial endotoxin in the sample by using a LAL assay. 